The invention relates to a latex enhanced immunoturbidimetry kit for measuring the content of C peptide in human serum, a preparation method of the kit and a method for detecting the content of C peptide in human serum by use of the kit. The kit comprises a first reagent, a second reagent and a calibrator, wherein the first reagent contains buffer solution, electrolyte, a surface active agent, a stabilizing agent, a reaction enhancer and a preservative; the second reagent contains buffer solution, electrolyte, a surface active agent, a preservative and anti-human C peptide antibody enveloped sensitization polystyrene latex particles; and the calibrator contains buffer solution, a surface active agent, a stabilizing agent, a preservative and human C peptide protein. Compared with the existing kit used clinically, the kit provided by the invention has strong specificity, high sensitivity and high accuracy, the kit is suitable for biochemical analyzers of common detection mechanism, has short detection time, can be operated conveniently, has low detection cost, has no radioactive contamination, and is suitable for popularization and application clinically.