The invention provides a method for measuring sialic acid content of protein. The method comprises the steps of 1, using sialidase to conduct digestive treatment on a protein sample, so that digestive juice containing sialic acid is obtained; 2, conducting sedimentation treatment on the digestive juice, and separating supernatant containing sialic acid; 3, using an ultra-high performance liquid chromatography-mass spectrometry method to analyze the supernatant, so that a mass spectrogram is obtained; 4, based on the mass spectrogram, determining the sialic acid content of protein. In the method for measuring the sialic acid content of protein, on the condition that there is no need to conduct derivatization on sialic acid, the total sialic acid content in the protein sample is subjected to rapid quantitative detection, the method is easy to operate, the sensitivity is high, the accuracy is high, the elapsed time is short, and the method can be used for intermediate quality control over biomacromolecule production or quality inspection of final products.