Abstract: A method based on immunoaffinity column clean-up and reversed-phase highperformance liquid chromatography (RP-HPLC) with fluorescence detection has been applied to determinate aflatoxins B1, B2, G1, G2 and M1 from spiked cheese. Chromatographic conditions were bettered by gradient elution technique. Calibration curves of aflatoxins G1, B1 and M1 were linear up to 2～20μg/L, and the calibration curves of aflatoxins G2 and B2 were linear up to 0.6～6.0μg/L. Limits of determination of B1, B2, G1, G2 and M1 were 0.2, 0.3, 0.06, 0.1 and 0.4μg/L, respectively. Average recoveries of the aflatoxins B1, B2, G1, G2 and M1 from cheese spiked with three different concentration levels were greater than 76%, and the RSDs ranged from 1.7% to 4.5%.