An HPLC method was established for determination of amino acids in human prothrombin complex.Each kind of amino acids reacted with 6-aminoquinolyl-N-hydroxysuccinimdyl-carbamate (AQC) resp., and their corresponding derivatives were analyzed and quantified by the Agilent 1200 HPLC using AccQ-Tag C18 column (waters 150 mm×3.9 mm, 4 μm) for gradient elution, with the mobile phase of water, eluent diluent (acetic acid salt-phosphate buffer) and acetonitrile at a wavelength of 248 nm by UV-detector, the column temperature was set at 37°C, and the sample volume was 10 μL.The amino acids were completely determined in 32 min, the recoveries were 99.3%-100.6%, and RSD was less than 1.5%.The method is simple, accurate and suitable for the determination of amino acids in human prothrombin complex.