Ružička was born in Vukovar, Croatia, then part of Kingdom of Hungary, Austro - Hungarian Empire. His family of craftsmen and farmers was mostly of Croatian origin, with a Czech great grandparent, and a great grandmother and a great grandfather from Austria.

Ružička attended the classics program secondary school in Osijek. He changed his original idea of becoming a priest and switched to studying technical disciplines.Chemistry was his choice, probably because he hoped to get a position at the newly opened sugar refinery built in Osijek.

Due to the excessive hardship of everyday and political life, he left and chose the High Technical School in Karlsruhe in Germany. He was a good student in areas he liked and that he thought would be necessary and beneficial in his future, which was organic chemistry. That is why his physical chemistry professor, Fritz Haber (Nobel laureate in 1918), opposed his summa cum laude degree. However, in the course of his studies, Ružička set up an excellent cooperation with Hermann Staudinger (a Nobel laureate in 1953). Studying within Staudinger's department, he obtained his doctor's degree in 1910. With Staudinger, Ružička went to Zurich and was his assistant.

Ružička's first works originated during that period in the field of chemistry of natural compounds. He remained in this field of research all his life. He investigated the ingredients of the Dalmatian insect powder (Pyrethrum cinerariifolium), a highly esteemed insecticide. In this way, he came into contact with the chemistry of terpene, a fragrant oil of vegetable origin, interesting to the perfume industry. He intended to start individual research and even started successful and productive cooperation with the Chuit & Naef Company (later known as Firmenich) in Geneva.

In 1916 - 1917, he received the support of the oldest perfume manufacturer in the world Haarman & Reimer, of Holzminden in Germany. With expertise in the terpene field, he became senior lecturer in 1918, and in 1923, honorary professor at the ETH (Eidgenössische Technische Hochschule) as well the University in Zurich. Here, with a group of his doctoral students, he proved the structure and existence of the compounds of muscone and civet, the scents derived from the musk deer and the civet cat. The Ruzicka large ring synthesis is a method inorganic chemistry for the organic synthesis of these type of compounds.

In 1921, the Geneva perfume manufacturers Chuit & Naef asked him to collaborate. Working here, Ružička achieved financial independence, but not as big as he did plan so he left Zurich to start working for the Ciba, Basel - based company. In 1927, he took over the organic chemistry chair at Utrecht University in Netherlands. In Netherlands he remained for three years, and then returned to Switzerland, which was superior in its chemical industry.

Back to Zurich, at ETH he became professor of organic chemistry and started the most brilliant period of his professional career. He widened the area of his research, adding to it the chemistry of higher terpenes and steroids. After the successful synthesis of sex hormones (androsterone and testosterone), his laboratory became the world center of organic chemistry.

In 1939, he won the Nobel prize for chemistry with Adolf Butenandt. In 1940, following the award, he was invited by the Croatian Chemical Association, where he delivered a lecture to an over packed hall of dignitaries. The topic of the lecture was From the Dalmatian Insect Powder to Sex Hormones. During the World War II, some of his excellent collaborators were lost, but Ružička restructured his laboratory with new, younger and promising people; among them was young scientist Vladimir Prelog. With new people and ideas new research areas were opened.

Following 1950, Ružička returned to chemistry, which had entered a new era of research. Now he turned to the field of biochemistry, the problems of evolution and genesis of life, particularly to the biogenesis of terpenes. He published his hypothesis, Biogenetic Isoprene Rule, which was the peak of his scientific career. Ružička retired in 1957, turning over the running of the laboratory to his assistant and future Nobel laureate Vladimir Prelog.

Ružička dedicated significant efforts to the problems of education. He insisted on a better organization of academic education and scientific work in the new Yugoslavia, and established the Swiss - Yugoslav Society. Ružička became an honorary academician at the then Yugoslav Academy of Sciences and Arts in Zagreb. In Switzerland, the Ružička Award was established, for young chemists working in Switzerland. In his native Vukovar, a museum was opened in his honour in 1977.

Ružička married twice: to Anna Hausmann in 1912, and 1951 to Gertrud Acklin. He died in Mammern, Switzerland, a village on Lake Constance.