Lysenko was born in Hrynky, Kremenchuk Povit, Poltava Governorate, the son of Vitaliy Romanovich Lysenko (Ukrainian: Віталій Романович Лисенко). From childhood he was very interested in the folksongs of Ukrainian peasants and in the poetry of Taras Shevchenko. When Shevchenko's body was brought to Ukraine after his death in 1861, Lysenko was a pallbearer. During his time at Kiev University, Lysenko dedicated himself to collecting and arranging Ukrainian folksongs, which were published in seven volumes. One of his principal sources was the kobzar Ostap Veresai (after whom Lysenko later named his son).

Lysenko initially studied Biology at the Kharkiv University, studying music privately. On a scholarship given to him by the Russian Music Society he pursued further professional music studies at the Leipzig Conservatory. It is there that he understood the importance of collecting, developing and creating Ukrainian music rather than duplicating the work of Western classical composers.

On his return to Kiev he began the creation of Ukrainian themed compositions. His Ukrainophilic approach to composition was not supported by the Russian Imperial Music Society which promoted a Great Russian cultural presence in Ukraine. As a result Lysenko severed his relationship with them, never to compose any music set to the Russian language, nor allow any translations of his works into Russian.

In order to improve his orchestration and composition skills the young Lysenko traveled to St. Petersburg where he took orchestration lessons from Nikolay Rimsky - Korsakov in the mid 1870s, but his fervent Ukrainian nationalism and disdain for Russian autocracy retarded his career. He supported the 1905 revolution and was in jail briefly in 1907. In 1908, he was the head of the Ukrainian Club, an association of Ukrainian national public figures in Kiev.

For his opera libretti Lysenko insisted on using only the Ukrainian language. Tchaikovsky was impressed by Lysenko's Taras Bulba and wanted to stage the work in Moscow, but Lysenko's insistence on it being performed in the Ukrainian language, not Russian, prevented the performance from taking place in Moscow.

In his later years, Lysenko raised funds to open a Ukrainian School of Music. His death was widely mourned throughout Ukraine. Lysenko's daughter Mariana followed her father's footsteps as a pianist, and his son Ostap also taught music in Kiev.

Lysenko's piano music is little known outside Ukraine. His piano works were considered by Soviet musicologists to be derivatives of Chopin. As a result they have not sparked as much interest to musicologists as his vocal music.

Lysenko's early vocal music consisted of settings of Ukrainian folk songs and compositions primarily to the poetry of Taras Shevchenko. Later he began to include other Ukrainian poets in his portfolio and also translations of works by German poets, some of which he did himself. Interest in Lysenko's art songs to the words of prominent Ukrainian poets such as Taras Shevchenko, Lesia Ukrainka, and Oleksander Oles is increasing, partly due to the recent efforts of the British opera singer Pavlo Hunka.

Lysenko wrote a number of operatic works, including Natalka Poltavka, Utoplena (The Drowned Woman, after Gogol's May Night) and Taras Bulba.

Lysenko made the first ethno - organological studies of the music of the blind kobzar Ostap Veresai which he published in 1873 - 4; they are still exemplary. In this work Lysenko demonstrated the way in which Ukrainian melodic material differs from Russian analogues by its unique use and approach to chromaticism (something that was censored out in Soviet republications). Lysenko continued to research and transcribe the repertoire of other kobzars from other regions such as Opanas Slastion from Poltava and Pavlo Bratytsia from Chernihiv. He also made a thorough study of other Ukrainian folk instruments such as the torban. His collection of essays about Ukrainian folk instruments makes him the founder of Ukrainian organology.