Abstract： Objective To study the intraocular penetration and pharmacokinetics of amphotericin B and rifampicin following topically applying compound amphotericin B eye gel in rabbit eyes. Methods Thirty rabbits were randomly divided into 10 groups. The tears were collected at 5, 15, 30, 45, 60, 90, 120, 180, 240 and 360 minutes after compound amphotericin B eye gel was applied to the rabbit eyes. Then the rabbits were sacrificed and the cornea and aqueous humors were collected immediately. The concentrations of amphotericin B and rifampicin were detected by High-performance liquid chromatography (HPLC) and pharmacokinetic parameters were estimated.Results Rifampicin could rapidly penetrate into rabbit ocular tissues, but amphotericin B in aqueous humors could not be detected by HPLC. Amphotericin B could maintain high concentration in tears for 4 hours. The pharmacokinetic parameters of amphotericin B and rifampicin in rabbits cornea were as follows: Tpeak was 20.96 and 7.57 min,Cmax was 3.32 and 25.37 μg/g, t1/2 was 45.34 and 31.69 min and AUC was 299.44 and 1368.91 min ·μg/g.Conclusions The results may provide experimental data for treatment of extraocular fungal infectious disease with compound amphotericin B eye gel. However, the therapeutic effect of compound amphotericin B eye gel on intraocular fungal infection need to be furtherly studied.