The invention discloses a bioinformatics method based on a protein mass spectrum data annotation eukaryote genome. The method specifically comprises the steps of 1, constructing a high-coverage eukaryote multi-omics sequence database; 2, removing eukaryotic protein sequence database redundancy; 3, conducting mass spectrum original data format conversion; 4, adopting a database searching engine with different algorithms, and retrieving mass spectrum data separately; 5, conducting peptide fragment spectrum matching and scoring on a retrieved and processed result; 6, screening result data after type FDR system evaluation; 7, verifying an annotated encoding gene; 8, authenticating a new gene which is not annotated; 9, authenticating alternative splicing; 10, authenticating functional point mutation; 11, aiming at protein posttranslational modification, conducting large-scale authentication; 12, conducting functional annotation of the new gene and the posttranslational modification. According to the bioinformatics method based on the protein mass spectrum data annotation eukaryote genome, the accuracy and sensitivity of protein mass spectrum data analysis are comprehensively improved, in-depth analysis and annotation for the eukaryote genome is achieved, and the method specifically has the advantages of being efficient, accurate and comprehensive.