OBJECTIVE： To establish a method for the determination of antibacterial effect of Ofloxacin eye drop. METHODS： According to the requirements in Chinese Pharmacopoeia （2015 edition） “antibacterial effect test”， using Staphylococcus aureus， Pseudomonas aeruginosa， Escherichia coli， Aspergillus niger and Candida albicans as test bacteria， colonycount method suitability test was conducted for Ofloxacin eye drop samples， the verified method was used to determine the number of viable bacteria at each time point， calculate the bacteria number of 1 mL in the sample and the number of bacteria at each time point and convert to lg value. RESULTS： Ofloxacin eye drop sample can reach the standard of antibacterial effectiveness of the “B”. CONCLUSIONS： The method can effectively determine whether the antibacterial effect of Ofloxacin eye drop fits the standard.