In this paper, related substances in enrofloxacin, including ciprofloxacin, 6-ethylpiperazinyl-7-chloro-enrofloxacin and defluorinated enrofloxacin, were detected by HPLC. The results showed that there was no 6-ethylpiperazinyl-7-chloro-enrofloxacin detected, and the quality of enrofloxacin could be controlled by the method in Europe Pharmacopeia.