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 Hg-2(RS)(2)). The vary in the peak current of Hg-2(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.