Objective: A new technical idea was provided for the study of biogenic amine formation mechanism and food safety risk assessment. Methods: An analytical method was established for determination of tyramine, phenylethylamine, tryptamine, octopamine and cadaverine in cheeses with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Biogenic amines were extracted by ultrasonic-assisted solvent extraction from cheeses, and were separated by HILIC column, determined by positive electrospray ionization (ESI) with multiple reaction monitoring (MRM) mode, quantified by the internal standard method. The effect of extraction solvent type, ultrasonic time and matrix effect on the peak area was studied, and the performance of the method was validated and evaluated. Results: Using 0.1% formic acid (acetonitrile/water) as the extraction solvent, the samples were extracted by ultrasound for 15 min. Ammonium acetate of 5 mmol/L (pH value was adjusted to 4 by formic acid) and acetonitrile were used as the mobile phase，biogenic amines without derivatization were detected by HPLC-MS/MS. The recoveries of 5 biogenic amines were 78.6%～113.8% and relative standard deviations were 2.41%～7.12% at three spiked levels. The limit of detection of tryptamine, phenylethylamine, tyramine and octopamine was 0.75 μg/kg and the limit of quantification was 2.5 μg/kg; The limit of detection of cadaverine was 1.50 μg/kg and the limit of quantification was 5.0 μg/kg. The linear relationship was good in the respective linear ranges, and the correlation coefficient was greater than 0.99. The method was used for the determination of commercially available cheese samples. The contents of different biogenic amines in different samples varied greatly, ranging from 4.45 to 2 863.00 μg/kg, which was within the safe range. Conclusion: The proposed method was suitable for fast determination of 5 kinds of biogenic amines in cheese.