Abstract: A method of HPLC-MS/MS was proposed for the determination of natamycin in food. The sample was extracted with a mixture of methanol and acetic acid by ultrasonic,and the extract was separated on a Dikma Endeavorsil C18 column, using a mixture of methanol and 0.3% acetic acid as mobile phase for isocratic elution. ESI+ and MRM were adopted in MS/MS.The linear relationship between the peak area and the mass concentration of natamycin was in the range of 0.01-1.0 mg·L-1, with detection limit (3S/N) of 0.05 mg·kg-1. The recovery rate measured by standard addition method was in the range of 79.9%-97.3%, with RSD (n=5) in the range of 4.0%-6.1%.