The invention provides a methotrexate latex immunoturbidimetry kit and a preparation method thereof. The kit is used for determining the content of plasma drug methotrexate in human serum, and is composed of a reagent R1 and a reagent R2 in a ratio of 1 to 1. R1 is prepared from the following components by weight percentage: 0.01% to 4% of an emulsifier, 0.01% to 4% of a methotrexate conjugate, 0.1% to 2% of sodium chloride, 0.05% to 2% of a sensitizer, 0.02% of a preservative, and a 30 to 100 mmol / L buffer. R2 is prepared from the following components by weight percentage: 0.1% to 0.5% of latex particles combined with an anti-methotrexate antibody, 0.1% to 1.0% of bovine serum albumin, 0.02% to 2% of Tween 20, 0.02 % to 2% of a stabilizer 2, 0.02% to 2% of a protein protectant 2, 0.02% ofpreservative, and a 20 to 50 mmol / L buffer. The kit provided by the invention has high sensitivity and stability, and has a good clinical application prospect, and the invention provides the preparation method.