Aspergillus oryzae, a filamentous fungus, can secrete large amounts of enzymes extracellularly. We constructed a genetically engineered A. oryzae that simultaneously produced cellobiohydrolase, endoglucanase, and beta-glucosidase by integrating multiple copies of the genes encoding these cellulases into fungal chromosomes. The resulting strain possessed 5-16 copies of each cellulase gene within the chromosome and showed approximately 10-fold higher activity versus single integration strains. Copy number polymorphisms were attributed to differences in flanking region sequence for the integrated gene fragments. Furthermore, we found that the P-sodM/T-glaB set demonstrated the strongest transcription levels per gene copy number. We therefore modified promoter/terminator set and cellulase gene combinations based on this polymorphism and transcription level data, with the resulting transformant showing 40-fold higher cellulolytic activity versus the single integration strain. This designed expression method could be useful for the overexpression of multiple enzymes and pathway flux control-mediated metabolic engineering in A. oryzae.