The present invention provides a pyruvic acid determination reagent box. The reagent box comprises a reagent R1 and a reagent R2. The reagent R1 comprises the following ingredients: reduced coenzyme I(NADH), a trihydroxymethyl aminomethane (Tris) buffer solution, a surface active agent, EDTA-Na2 and a preservative. The reagent R2 comprises the following ingredients: the trihydroxymethyl aminomethane (Tris) buffer solution, lactate dehydrogenase (LDH), the surface active agent, a stabilizer and the preservative. The present also provides a preparation method of the reagent box and applications. The reagent box is a high-stability, high-accuracy and good-repeatability liquid reagent box.