With 3, 3’5, 5’-tetramethylbenzidine(TMB) as the detection substrate, a reliable and highly selective method was established and optimized for the determination of Lactoperoxidase(LP) activity in raw milk. The method was based on the enzymatic reaction principle, where hydrogen peroxide oxidated TMB in the presence of LP. The optimized conditions of this assay system were obtained, consisting of 20 mmol · L~(-1) TMB solution, 0.6 mmol · L~(-1) hydrogen peroxide and 0.1 mol · L~(-1) Citric Acid(CA)/0.2 mol · L~(-1) disodium hydrogen phosphate(Na P) buffer(pH 4.8). TMB detection method was applied to the analysis of LP in milk samples with a practical working concentration range from 2 to 14 mg · L~(-1). The intra-and inter-batch variation coefficients were all below 5%, indicating a good repeatability. Confirmation test between TMB method and 2, 2-azinobi(3-ethylbenzothiazoline-6-sulphonate) diammonium salt(ABTS) method was carried out, and the results of TMB assay were in accordance with that of ABTS method. With 3, 3’5, 5’-tetramethylbenzidine (TMB) as the detection substrate, a reliable and highly selective method was established and optimized for the determination of Lactoperoxidase (LP) activity in raw milk. The method was based on the enzymatic reaction The optimal conditions for this assay system were obtained as follows: TMB solution, 20 mmol · L -1 TMB solution, 0.6 mmol · L -1 hydrogen peroxide and 0.1 mol · L -1 Citric Acid (CA) /0.2 mol · L -1 disodium hydrogen phosphate (Na P) buffer (pH 4.8). TMB detection method was applied to the analysis of LP in milk samples with a practical working concentration range from 2 to 14 mg · L -1. The intra-and inter-batch variation coefficients were all below 5%, indicating a good repeatability. Confirmation test between TMB method and 2, 2-azinobi ( 3-ethylbenzothiazoline-6-sulphonate) diammonium salt (ABTS) method was carried out, and the results of TMB assay were in accordan ce with that of ABTS method.