Abstract: To investigate the effect of various protease treatments on the aroma-active compounds in Agrocybe aegerita, proteases, including flavourzyme, protamex, neutral protease, and trypsin were used to hydrolyze Agrocybe aegerita, and the flavor profiles of the resulting hydrolysates were evaluated through sensory analysis. Additionally, electronic nose (e-nose) technology and solid-phase microextraction coupled with comprehensive two-dimensional gas chromatography-olfactometry-time-of-flight mass spectrometry (SPME-GC×GC-O-TOF/MS) were employed to analyze the aroma-active compounds in the protease-treated samples. The results showed that enzymatic hydrolysis significantly influenced both the flavor profile and the composition of aroma-active compounds in Agrocybe aegerita. Sensory evaluation showed that, following enzymatic treatment, the intensity of mushroom, pungent, malt, and buttery flavors was reduced, while fruity, floral, and nutty aromas became more pronounced. A total of 50, 57, 52, 52, and 48 aroma-active compounds were identified in the control and the four protease-treated hydrolysates, respectively. These compounds, including alcohols, esters, aldehydes, ketones, and pyrazines, collectively contributed to the distinctive aroma of Agrocybe aegerita and its hydrolysates. The sensory results further confirmed the observed changes in aroma-active compounds, such as the increased presence of ketones, pyrazines, and nitrogen-, oxygen-, and sulfur-containing compounds, alongside a reduction in aldehydes with pungent odors. These changes were key factors contributing to the enhanced flavor of the hydrolysates compared to the control. Among the treatments, the hydrolysate derived from flavourzyme exhibited the most favorable flavor profile. Enzymatic hydrolysis not only preserved but also improved the flavor of Agrocybe aegerita, and provided valuable scientific evidence for optimizing its flavor characteristics and offering new theoretical insights for its potential applications in the food industry.