This research aims to investigate synthesis parameters for modification of chitosan with enrofloxacin. Most modifications of chitosan are focused on amino group. There are many methods for modification of chitosan, for example, conjugation of amino group with carboxylic group by coupling agents. 1-Ethyl-3-(3-(dimethylamino) propyl) carbodiimide hydrochloride (WSC) and N-hydroxysuccinimide (NHS) are widely used coupling agents for amide formation under mild conditions. Those two coupling agents were applied in this study. Optimum amount and ratio of coupling agents, amount of enrofloxacin and reaction times were investigated. The chemical structures were characterized by Fourier transform infrared spectroscopy (FT-IR). The optimum synthesis conditions were determined from the absorbance ratios. Chitosan-enrofloxacin has antibacterial activity against both S. aureus and E. coli.