【Objective】 To determine the optimum conditions for total sialic acid analysis in serum using the fluorometric high-performance liquid chromatography (HPLC). 【Methods】 The sialic acid standard operating fluid contained N-acetylneuraminic acid (Neu5Ac),N-glycolylneuraminic acids (Neu5Gc) and ketodeoxynonulosonic acid (KDN).The human plasma would be hydrolyzed with the trifluoroacetic acid (TFA),sulphuric acid and hydrochloric acid at different concentration of 0.05,0.10,0.15 mol/L and 0.20 mol/L at 80 ℃ and at varying time of 30,60,90 min and 120 min respectively.The hydrolyzed sample was treated with 4,5-Methylenedioxy-1,2- phenylenediamine dihydrochloride (DMB,Sigma) derivative reagent at 50 ℃ for 150 min away from light. 【Results】 The maximum release of sialic acid was obtained with an equal volume of 0.1 mol/L TFA for 60 min at 80 ℃.The optimum detection limit of sialic acid was in 0.625~80 μmol/L,the average recovery rate was 94.0% and the relative standard deviation (RSD) for the precision,stability and repeatability test were 1.3%,1.1% and 1.0%,respectively.The minimum detection limit of Neu5Ac was 0.02 μmol/L and the average content of sialic acid in human serum was (2.12±0.15) mmol/L in 16~19 years old healthy adolescents. 【Conclusion】 The total sialic acid content determinated with fluorometric HPLC method is a simple,high sensitivity and good repeatability,which can be widely used in determination of sialic acid concentration in serum.