The invention discloses an Escherichia coli expression system for producing oxalate oxidase, a production method of oxalate oxidase and application thereof. The recombinant expression plasmid in the E. coli expression system comprises: an oxalate oxidase gene, a chaperone gene, and a gene that promotes to pump manganese ions into E. coli cell-associated protein. By screening and testing the genesof different manganese ion-related proteins and their copy number, the type of a promoter for controlling expression of the molecular chaperone and the type of a culture medium used in the productionprocess of oxalate oxidase, an optimized expression combination of oxalate oxidase is obtained, and the expression activity of oxalate oxidase in E. coli exceeds 80 U/mL. The specific activity of theoxalate oxidase preliminarily purified by a supernatant of a cell disrupted liquid is greater than 20 U/mg, and the molecular weight of the monomer is about 25 kDa. The technical scheme of the invention has the advantages of simple production and purification process, high expression level and specific activity, easy industrial amplification and low cost, and is advantageous for industrial production and application of such enzymes.