This study analyzed the effects of different nitrogen sources in substrate composition on the growth of Pleurotus ostreatus, as well as the subsequent changes in flavor and antioxidant activity. The T2, composed of poplar sawdust, beet pulp, cotton seed dregs, and cotton seed coat in a ratio of 40:20:20:20, exhibited the highest yield at 156.6 g. The total polyphenol content and ABTS and DPPH radical scavenging activities were 8.25 mg GAE/g, 70%, and 49%, respectively, showing higher radical scavenging activity compared to the Control and T1. Additionally, varying nitrogen content resulted in distinct aroma patterns and is presumed to influence taste profiles such as sourness, umami, and saltiness.