The invention provides a cystatin C detection kit and a preparation method thereof. The kit comprises a reagent R1, a reagent R2 and a cystatin C calibration product, wherein the reagent R1 comprisesa preservative, a surfactant, an emulsifier and a buffer solution; the reagent R2 comprises a cystatin C antibody, polystyrene latex microspheres, a latex microsphere activating agent, a sealing agent, an emulsifier, a preservative, a surfactant and a buffer solution. The cystatin C antibody is slowly dropwise added to an activated polystyrene latex microsphere solution, a polystyrene latex microsphere suspension coupled with the cystatin C antibody is obtained after a stirring reaction, sealing is performed, the antibody and latex microspheres are coupled together in a chemical crosslinking manner, and centrifugation, cleaning and ultrasonic resuspension processes in conventional methods are omitted. The production process is simplified, and the kit has the advantages of low blank absorbency, high sensitivity and precision and large linear range.