The invention discloses a preparation method for a sing-component TMB color development solution. The preparation method comprises the followings steps: respectively weighting 7-10 g of citric acid, 16-20 g of sodium hydrogen phosphate and 0.1-0.5 g of perboric acid soda to be placed in a 1000 mL beaker A, and adding into 800 mL of purified water to enable the mixture to be dissolved; secondly, adjusting the pH of the solution in the beaker A to be 4.5-6.0, adding into 20-30 mL of glycerol, and then adding into 1-3 g of EDTA; thirdly, weighing 5-40 mg of TMB to be placed in a 1000 mL beaker B, and adding into 10-30 mL of alcohol to enable TMB to be dissolved; finally, transferring the solution in the beaker A and the solution in the beaker B to a 1000 mL volumetric flask, adding purified water to set the volume to be 1000 ML, and shaking. Compared with a conventional two-component TMB color development solution, the single-component TMB color development solution prepared by the invention has the advantages of being convenient to operate, high in sensitivity, good in stability and low in color development background.