The invention provides a kit for measuring the content of retinol-binding protein in human serum and a preparation method thereof. Reagent R1 and reagent R2 are composed of 3:1 volume ratio, R1: 0.01% to 4% w / w emulsifier, 0.1% to 2% w / w sodium chloride, 0.05% to 2% w / w Sensitizer, 0.02% w / w preservative, 30-100mmol / L buffer. R2: 0.1%-0.5% w / w latex microparticles bound with anti-human retinol-binding protein antibody, 0.1%-1.0% w / w bovine serum albumin, 0.02%-2% w / w Tween 20, 0.02 %-2% w / w stabilizer 2, 0.02%-2% w / w protein protectant 2, 0.02% w / w preservative, 20-50 mmol / L buffer. The kit of the invention has wide linear range, good sensitivity and stability, and has good clinical application prospect.