Wislicenus was born in Klein-Eichstadt, Germany, the son of a Lutheran pastor who was forced to flee Europe in 1853 because of his political views. Wislicenus accompanied his father to America, where he attended Harvard until returning to Europe in 1856. He continued his education at Halle and at Zurich, where he was appointed professor of chemistry in 1870. In 1872 he moved to Würzburg, where he stayed until he succeeded Adolph Kolbe as professor of chemistry at the University of Leipzig (1885).

In 1872 Wislicenus showed that there were two forms of lactic acid having the formula CH₃CH(OH)COOH. One, derived from sour milk by Carl Scheele in 1870, was optically inactive; the other, discovered by Jöns Jacob Berzelius in 1808, was active. Wislicenus suggested that this was caused by different arrangements of the same atoms in space producing different properties in the compounds. Wislicenus's findings and similar work led Jacobus van't Hoff and Joseph Le Bel to establish the new discipline of stereochemistry a few years later. Wislicenus went on to study ‘geometrical isomerism’ – the existence of isomers because of different arrangements of groups or atoms about a double bond in the molecule.