Abstract: The cell wall proteinase gene(prtB)was amplified by PCR using the Lactobacillus bulgaricus genome as a template,and the comparison and bioinformatics analysis of prtB were performed using GeneBank database and biological software to predict the properties of gene fragments,such as similarity,expression,protein size,secondary structure and biofilm binding. Results showed that,it was 100% similar to the prtB of template Lactobacillus bulgaricus after sequencing,which proved that the gene fragment did not undergo mutation. prtB showed 96% identity with Lactobacillus delbrueckii subsp. delbrueckii gene,and 82% identity with Lactobacillus delbrueckii subsp. lactis,which indicated the fragment had certain conservatism and specificity. The molecular weight of prtB was 3338.7 kD,and the contents of G+C and A+T were 47.49% and 52.51%. The prtB encoded 1831 amino acids,the protein of which was named PrtB,with isoelectric point pI=8.76. The protein PrtB as a hydrophilic stable protein had a total average hydrophilicity of-0.583,28 Ser,9 Thr and 25 Tyr as the possible site of protein kinase phosphorylation,which should be extracellular protein. The secondary structure of PrtB was mainly composed of irregular curl up to 55.11%,21.30% alpha helix and 23.59% beta fold.