At first John attended the public school gymnasium in Mühlhausen. Recognizing his intelligence at a young age, Roebling's mother, Friederike Dorothea Roebling arranged for him to be in mathematics and science at Erfurt close to Ephraim Solomon Unger. He went to Erfurt when he was 15. In 1824, he passed his Surveyor's examination and returned home for a year. In 1824 he enrolled for two semesters at the Bauakademie in Berlin, where he studied architecture and engineering under Martin Friedrich Rabe (1765 – 1856), bridge construction and foundation construction under Johann Friedrich Dietlein (1782 – 1837), hydraulics under Johann Albert Eytelwein (1764 – 1848), and languages. Roebling also attended lectures of philosopher Georg Hegel. Roebling developed an interest in natural philosophy and many years later he worked on a 1000 page treatise about his own concepts of the universe.

In 1825 he got a government job at Arnsberg, Westphalia, working on military road building for four years. During this period he made sketches for suspension bridges. In 1829 he returned to his home to work out his final thesis and prepare for his second engineers' examination. For unknown reasons, he never took the examination.

On May 22, 1831, Roebling left Germany with his brother Karl and visionary Johann Adolphus Etzler. Economic mobility and career advancement were difficult for engineers in Prussian society. This unfortunate state of affairs had been brought about by the Napoleonic Wars, which lasted until 1815. This period in European history left Prussia with political unrest, as authoritarian governments traded places with democratic ones. Etzler had ideas about creating a utopia in the United States, but disputes arose en route, and the group split. John and Karl purchased 1582 acres (6.4 km²) of land on October 28, 1831, in Butler County, Pennsylvania with the intent to establish a German settlement, called Saxonburg. Most of the other settlers remained with Etzler.

John Roebling could not have arrived in the United States at a worse time. The nation was gripped by an eighty year financial depression. Farmers were deeply affected by it. A dominant mode of thought in America was manifest destiny, and the opening of the East, and transportation between eastern industrial hubs and frontier farming markets had become a matter of both national and popular interest. Many transportation projects were underway near the location he chose for his colony, but instead of continuing an engineering profession, he took up farming. After five years he married Johanna Herting Roebling, a tailor's daughter, and had nine children with her over the next decade.

Agrarian work was unsatisfactory to John Roebling, and the colony attracted very few settlers. In 1837, after the death of his brother and the birth of his first child, he returned to engineering as a vocation.

Roebling's first engineering work in America was devoted to improving river navigation and canal building. He spent three years surveying for railway lines across the Allegheny Mountains, from Harrisburg to Pittsburgh, for the state of Pennsylvania. In 1840, he wrote to suspension bridge designer Charles Ellet, Jr., offering to help with the design of a bridge near Philadelphia:

The study of suspension bridges formed for the last few years of my residence in Europe my favourite occupation ... Let but a single bridge of the kind be put up in Philadelphia, exhibiting all the beautiful forms of the system to full advantage, and it needs no prophecy to foretell the effect which the novel and useful features will produce upon the intelligent minds of the Americans.

In 1841, at Saxonburg, he began producing wire rope. During this time, canal boats from Philadelphia often were transported over the Allegheny Mountains on railroad cars to access waterways on the other side of the mountains, so that the boats could continue to Pittsburgh. The system of inclines and levels that moved the boats and conventional railroad cars was a state owned enterprise, the Allegheny Portage Railroad. The railroad cars were moved up and down the inclines by a long loop of hemp rope up to nine inches in circumference. The hemp ropes were very expensive, And rendered poor service life. Roebling remembered an article he read about wire ropes. Soon after, he started developing a 7-strand wire rope at a ropewalk he created on the meadow of his farm.

In 1844, Roebling won a bid to replace the wooden canal aqueduct across the Allegheny River with the Allegheny Aqueduct. His design encompassed seven spans of 163 feet, each consisting of a wooden trunk to hold the water supported by a continuous cable made of many parallel wires, wrapped tightly together, on each side of the trunk. This was followed in 1845 by building a suspension bridge over the Monongahela River at Pittsburgh. In 1848, Roebling undertook the construction of four suspension aqueducts on the Delaware and Hudson Canal. During this period, he moved to Trenton, New Jersey.

Roebling's next project, starting in 1851, was a railroad bridge connecting the New York Central and Great Western Railway of Canada over the Niagara River, which would take four years. The bridge, with a clear span of 825 feet, was supported by four, ten inch wire cables, and had two levels, one for vehicles and one for rail traffic. While the Niagara bridge was being built, Roebling undertook another railway suspension bridge, across the Kentucky River which required a clear span of 1,224 feet. The anchorage and stone towers were completed, and the cable wire delivered along with the material for the superstructure, when the railway company collapsed. The bridge was left uncompleted temporarily, to be later finished as a truss bridge.

In 1859, Roebling completed another suspension bridge at Pittsburgh, this one of 1,030 feet, divided into two spans of 344 feet each, and two side spans of 171 feet each.

The outbreak of the American Civil War brought a temporary halt to Roebling's work. During the war, in 1863, building resumed on a bridge over the Ohio River at Cincinnati which he had started in 1856 and halted due to financing, but the bridge was finished in 1867. The Cincinnati - Covington Bridge, later named after him John A. Roebling Suspension Bridge, would become the world's longest suspension bridge at the time of completion.

In 1867 Roebling started design work on what is now called the Brooklyn Bridge, spanning the East River in New York. One day in 1869 he was standing at the edge of a dock, working on fixing the location where the bridge would be built, when his foot was crushed by an arriving ferry. His injured toes were amputated. He refused further medical treatment and wanted to cure his foot by "water therapy" (continuous pouring of water over the wound). His condition deteriorated. He succumbed to tetanus 24 days after the accident.

Roebling devised “an equilibrium strength approach, in which equilibrium is always satisfied but compatibility of deformations is not enforced.” This was essentially an approximation method similar to the force method. First, Roebling found the dead and live loads. Roebling then divided the load between the cables and the stays. Roebling added a large safety factor to the divided loads and then solved for the forces. This approach gave a sufficiently accurate analysis of the structure given the assumption that the structure was ductile enough to handle such deformation.

Roebling's son Washington Roebling and his daughter-in-law Emily Warren Roebling continued his work on the Brooklyn Bridge. His 2nd son Ferdinand expanded his wire rope business. Roebling's 3rd son Charles Roebling designed and invented a huge 80 ton wire rope machine and founded the town of Roebling, New Jersey, where the John A. Roebling's Sons company steel mill was built. His grandson, Washington A. Roebling, II, died on the RMS Titanic. His great-grandson, Donald Roebling was a noted philanthropist and inventor who devised the amphtrack.