In this study, levofloxacin (LEVOF) hemihydrate interaction with L-cysteine (RSH) was investigated by using square-wave voltammetry (SWV) in Britton–Robinson (B–R) buffer pH 7.4. Addition of the LEVOF to RSH solution resulted in dropping of the main reduction peak current of RSH (the current of mercurous cysteine thiolate Hg2(RS)2). The vary in the peak current of Hg2(RS)2, after the adding of the LEVOF is indicated an interaction between RSH and LEVOF molecules. Moreover, the interaction between two molecules also confirmed with UV-Vis, FT-IR spectroscopic measurements and thermal analysis data. Binding constant of LEVOF with RSH was calculated by both voltammetric and UV-Vis spectroscopic data.