The invention discloses a protein C activity determination kit based on a chromophoric substrate method, protein C is a vitamin K dependent plasma serine protease zymogen, and comprises a reagent R1, a reagent R2 and a diluting reagent; the R1 reagent comprises a protein C activator, a first buffer solution and a first auxiliary material; the R2 reagent comprises a chromogenic substrate Pca-5297, a second buffer solution and a second auxiliary material; the diluting reagent comprises a third buffer solution and a third auxiliary material, and the pH of the first buffer solution, the pH of the second buffer solution and the pH of the third buffer solution are 7.2-7.6; the protein C activator and the chromophoric substrate Pca-5297 are matched for use, during detection, the cutting efficiency is high, the reaction signal is strong, the reaction speed is high, the sensitivity is high, the linear range is wide, the reaction time is short, the sample distinction degree is increased, and establishment of the linear range and clinical sample testing are facilitated; the reagent is liquid, convenient to use, low in cost and good in stability.