Aspergillus oryzae (A. oryzae) enzyme systems are critical for substrate utilization and flavor formation in soy sauce fermentation, which are key factors influencing soy sauce quality. To compare the differences in enzyme activity between two representative A. oryzae strains (AO-HP and AO-HC) and to screen for associated genetic variation, this study measured the activities of their protease, carbohydrate hydrolases, and γ-glutamyl transpeptidases. Additionally, whole-genome resequencing was performed using RIB40 as reference, to identify single nucleotide polymorphism (SNP), insertions or deletions (InDel), and copy number variation (CNV), followed by functional annotation and pathway enrichment analysis of candidate variant genes associated with key enzymes. The results showed that AO-HP exhibited higher activities of neutral protease, acid protease, and aminopeptidase, indicating stronger protein hydrolysis capacity, whereas AO-HC exhibited higher xylanase, glucoamylase, β-glucosidase, and γ-glutamyl transpeptidases activities, suggesting enhanced carbohydrate degradation and γ-glutamyl transfer potential. Genomic analysis revealed that AO-HC harbored more SNPs and InDels and showed a higher proportion of heterozygous variants compared with AO-HP. Variant genes were mainly enriched in carbohydrate metabolism and glycan biosynthesis and metabolism pathways. Nonsynonymous mutation screening identified 7 protease-related, 10 carbohydrate enzyme-related, and 3 γ-glutamyl transpeptidases-related candidate genes in AO-HC, compared with only 2 carbohydrate enzyme-related genes in AO-HP. These results suggest that strain-specific enzyme activity differences are associated with genetic variation in key enzyme-related genes, particularly those involved in carbohydrate metabolism and γ-glutamyl transfer in AO-HC. This study aims to provide a theoretical basis for functional gene mining and targeted breeding of industrial A. oryzae strains.