The invention discloses a method for measuring nucleotide content in a dairy product and belongs to the field of food additives and detection methods thereof. The method comprises the following steps: (1), preparing stock solutions of GMP, UMP, CMP, IMP, AMP, wherein the concentrations of the stock solutions are 1 mg/ml, and preparing into solutions with the series normal concentrations of 10.0 [mu]g/ml, 20.0 [mu]g/ml, 30.0 [mu]g/ml, 40.0 [mu]g/ml and 50.0 [mu]g/ml, wherein the sample size is 10 [mu]l, measuring the peak heights of the solutions through a liquid chromatograph and calculating a regression equation; (2), weighing 10.0 g of a sample, dissolving the sample with 20 ml of warm water, putting into an ultrasonic bath for oscillation for 10 min, then taking out, transferring into a 50-ml volumetric flask, adding acetic acid for protein precipitation, after diluting, uniformly mixing, filtering through filtering paper, filtering through a filtering membrane, and measuring the peak height of the sample through the high performance liquid chromatograph; (3), according to a standard curve equation and the peak height value of the sample, obtaining the nucleotide content in the dairy product. According to the invention, the liquid chromatography is adopted to measure the nucleotide content in an infant formula milk powder, the method is simple and convenient to operate, the measurement precision and accuracy can meet analysis requirements of the dairy product, and the method can be used as a normal inspection method.