Abstract: A method of online pre-column OPA-FMOC derivatization HPLC equipped with fluorescence detector were executed to determine the composition of amino acids. The results showed that 22 amino acids were separated in gradient elution within 14 min,and the calibration curves had good linear relationships within 5~200 nmol/mL. All of the coefficients of determination R²>0.998,detection limit 0.4~4 nmol/mL,recovery rates>80%. This method was successfully applied to quantitative determination of 22 amino acids of different sources. The method was simple and high-throughput,which could be used as a reference method for the rapid determination of amino acid content for the high protein food.