The invention relates to a method for detecting differentially-expressed protein. For quantitative proteomics, on the basis of marked and unmarked relative quantitative data analysis of primary spectrogram information, whether certain protein differentially expresses protein is judged according to corresponding spectrogram signal intensity ratio of the certain protein in two or more samples, and the method comprises the following steps: matching a peptide spectrum, evaluating credibility, extracting peptide fragment signals, calculating a peptide fragment ratio, calculating a protein ratio, and performing statistical analysis. For about 100 GB data collected through mass spectrum experiment, automated analysis is performed rapidly, for signals of different protein in a mass spectrometer, the protein signals can be extracted accurately as soon as possible, protein differential expression is determined from statistical significance, and accuracy of results is evaluated.