Abstract: Using genetic engineering technology,the recombinant strain TH2-protAS of Aspergillus niger which overexpressed the prolyl endopeptidase derived from Aspergillus niger with its own signal peptide and the glucoamylase signal peptide and α-Amylase signal peptide replaces its own signal peptide prolyl endopeptidase Aspergillus niger recombinant strains TH2-SglaA-protA and TH2-SamyA-protA. The results of SDS-PAGE showed that prolyl endopeptidase was secreted and expressed in the recombinant strain,but there were different degrees of glycosylation. The enzyme activity test results showed that the highest enzyme activities of recombinant strains TH2-protAS,TH2-SglaA-protA and TH2-SamyA-protA were 1.70,2.19,1.91 U/mL,respectively. On the basis of the recombinant strain TH2-SglaA-protA,gene knockout technology was used to knock out the main background protein acid-stable α-amylase,combined with optimization of fermentation conditions,to obtain high-purity prolyl endopeptidase. Based on the above results,it could conclude that prolyl endopeptidase could be secreted and expressed efficiently in Aspergillus niger. glaA signal peptide and amyA signal peptide significantly increased the secretion and expression of prolyl endopeptidase in Aspergillus niger,and the effect of signal peptide glaA was better than that of amyA signal peptide. The combination of gene knockout and fermentation regulation could effectively remove the secreted background protein and obtain a strain with high-yield and high-purity prolyl endopeptidase. This recombinant strain had a clear industrial application potential.