This study aimed to optimize endo-1,4-β-glucanase (endoglucanase) production by Aspergillus oryzae PTCC 5164 growing on wheat straw under solid-state fermentation (SSF), investigate fungal growth and enzyme production kinetics, and evaluate enzymatic hydrolysis of wheat straw for glucose release using concentrated cell-free enzyme solutions. A 2⁴ full factorial design was employed to assess the effects of particle size (0.3–2.0 cm), moisture content (65–85%), ammonium sulfate (0.04–0.12 g/g straw), and carboxymethyl cellulose (CMC, 0–0.08 g/g straw) on endoglucanase yield. In a separate experiment, fungal biomass, enzyme activity, and substrate consumption were monitored over seven days, and biomass yield on substrate (YX/S) and enzyme yield on biomass (YE/X) were calculated. Hydrolysis assays were performed on wheat straw using concentrated cell-free enzyme solutions at varying Straw loading (w/v)%. The results showed that moisture and particle size were the dominant factors influencing endoglucanase production, with significant higher-order interactions with CMC and ammonium sulfate. Maximum endoglucanase activity (9.93 IU/g straw) was achieved at the particle size of 0.3 cm, CMC concentration of 0.08 g/g straw, ammonium sulfate concentration of 0.12 g/g straw, and 65% moisture content. During the seven-day monitoring period, peak endoglucanase activity (6.88 IU/g straw, corresponding to a YE/X of 3.54 IU/mg biomass protein) was observed on day 3, while peak biomass (2.34 mg biomass protein/g straw, corresponding to a YX/S of 9.58 mg biomass protein/g consumed straw) was observed on day 5. Enzymatic hydrolysis of wheat straw released up to 5.43 mg glucose/mL, with Straw loading (w/v)% of 8%w/v and enzyme concentration of 0.5 IU/mL. Overall, these results establish that A. oryzae PTCC 5164 can efficiently convert wheat straw into cellulolytic enzymes under optimized SSF conditions, and the resulting enzyme preparation is effective for saccharification, offering a viable route for valorization of this agricultural residue.