In this paper a high-performance liquid chromatographic(HPLC) method has been developed to determine the concentration of tryptophan(Trp) in human serum. Serum sample was precipitated with trichloroacetic acid solution(0.6 mol.L-1) to remove protein and centrifuged and assayed by HPLC. The operating conditions were NOVA-PAK C18 column(3.9 mm x 150 mm), KH2PO4(0.1 mol.L-1) mobile phase with flow rate of 0.7 ml.min-1, and detective wave-length at 295 nm. The linear range was 1.223-2447.381 mumol.L-1. The detection limit was 0.612 mumol.L-1. The average recovery was 96.3%. Intra-assay CV is less than 4% and inter-assay less than 5%. The concentration of Trp in normal adult serum was 46.603 +/- 5.108 mumol.L-1. The detection time of serum Trp was within 5 min. The results suggest that this method is simple, fast, accurate, and convenient.