The invention discloses a lipase determination kit and a preparation method thereof. An R1 reagent comprises a buffer solution, sodium deoxycholate, colipase, metal salt, a protective agent, a surfactant and a preservative; an R2 reagent comprises Ltartaric acid, calculus bovis deoxycholic acid sodium salt, 1, 2-o-dilauroyl racemic glycerol 3valeric acid (6methyl resorufin) ester, a cosolvent, a stabilizer and a preservative, and the cosolvent is composed of ethyl acetate, dilauroyl glycerol sulfate and a Triton surfactant. According to the invention, through optimization, ethyl acetate, dilauryl glyceryl sulfate and Triton surfactant are jointly used as a cosolvent to play a synergistic effect of the three components, so that the solubility of a substrate is greatly improved, and further,mercaptoethanol and mannitol are used as composite stabilizers, therefore, the lipase determination kit which is good in stability, high in accuracy and precision and remarkably improved in linear range is obtained.