The invention provides a mass spectrometry detecting method for a serum polypeptides, comprising sample preparation, data-independent mass spectrometry acquisition, and data processing. According to the mass spectrometry detecting method for the serum polypeptides, a data-independent mass spectrometry acquisition method for serum peptidomics samples is developed, and the parent ion distribution ofthe sample by pre-analytical experiment on serum samples is obtained, thereby obtaining an optimal variable isolation window. Each condition and each step cooperate with each other by optimizing thepreparation method of the polypeptideomics sample, the mass spectrometry acquisition method, and the data processing method, and finally, the number of the detection of the polypeptide, the reproducibility of the identification, and the quantitative accuracy are significantly improved. The method of the invention has the characteristics of high throughput, high reproducibility, and high accuracy,and is suitable for analysis of large-scale clinical serum samples and detection of disease markers, thereby having wide application prospects and great market value.