Abstract: UHPLC was applied to the determination of aflatoxin M1 in milk. Sample was purified by immune affinity column and eluted from the column with acetonitrile. The eluate was separated on ACQUITY UPLC BEH C18 column (2.1 mm×50 mm, 1.7 μm) by gradient elution with mixtures of acetonitrile and water in different ratios as mobile phase, and determined by MS/MS. The positive electrospray ionization as well as multiple reactions monitoring mode was used in the detection. Linear relationship between values of peak area and mass concentration of aflatoxin M1 was obtained in the range of 0.1-16.0 μg·L-1. Values of limit of detection (3S/N) and lower limit of determination (10S/N) found were 0.001 5 μg·kg-1 and 0.005 μg·kg-1 respectively. The proposed method was used in the analysis of milk for its content of aflatoxin M1, giving values of recovery in the range of 89.5%-101.5% with RSD′s (n=6) in the range of 2.8%-9.5%.