The invention belongs to preparation of a new gene, construction of engineering bacteria, and particularly relates to a pyranose oxidase gene constructed from molecular chaperone, protein, phchia pastoris and preparation and application of the pyranose oxidase gene. Genes Ero I, PDI, CNE1, SEC53 and the like in pichia pastoris are respectively cloned and connected to series derivative plasmids ofpichia pastoris expression vectors pPICZ and the like for connection, and the genes and P20 are subjected to coexpression to realize conversion into Pichia pastoris GS115 bacterial strains to be coated to YPD flats being different in bleomycin concentration gradients for inspection of effects. Through overexpression of molecular chaperone and optimization of the fermentation condition, and throughscreening and identifying, a bacterial strain with enhanced secretory expression pyranose oxidase than an original bacterial strain is obtained. For the pyranose oxidase expressed by the bacterial strain, the enzyme activity in a 10L fermenter can achieve 405U/mL, and compared with the bacterial strain without overexpression of the molecular chaperone, the enzyme activity is improved by twice, sothat a favorable foundation is established for large-scale production of the pyranose oxidase.