The invention discloses an anti-interference one-component enzyme substrate color developing reagent. Each 1,000 mL of color developing liquid comprises the following components: 7-10 g of citric acid, 16-20 g of disodium hydrogen phosphate, 0.1-0.5 g of sodium perborate, 20-30 ml of glycerin, 5-40 mg of an enzyme substrate and 10-30 mL of ethanol; the pH value of the anti-interference one-component enzyme substrate color developing reagent is 4.0-6.0. By adjusting the pH value of the anti-interference one-component enzyme substrate color developing reagent to be 4.0-6.0, the anti-interference one-component enzyme substrate color developing reagent is relatively good in stability when being used for the color developing period in an enzyme-linked immunosorbent assay reaction, and has a relatively small color developing background; moreover, due to the one-component color developing liquid, the step of mixing liquid A with liquid B during use of the enzyme substrate color developing reagent is avoided, the operating time is shortened, the efficiency is improved, the concentration change of the effective color developing components of the enzyme substrate color developing reagent is avoided, and then the sensitivity of the enzyme substrate color developing reagent is improved.