With the increasing scarcity of traditional hardwood sawdust resources, developing sustainable substrates for edible fungi cultivation has become an urgent industrial priority. This study systematically evaluated the effects of bamboo sawdust substitutions (20%, 30%, 40%, and 50%) on mycelial growth, fruiting body development, and nutritional quality of Auricularia heimuer, while elucidating the underlying molecular mechanisms through transcriptome sequencing. The results demonstrated that bamboo substitution of ≤30% maintained normal mycelial growth and fruiting body differentiation, with 20% and 30% substitutions increasing yields by 5.30% and 3.70%, respectively, compared to the control. However, 50% substitution significantly reduced yield by 9.49%. Nutritional analysis revealed that 20–40% bamboo substitution significantly enhanced the contents of crude protein, polysaccharides, and essential minerals (calcium, iron, and selenium) in fruiting bodies. Transcriptome analysis identified upregulation of glycosyl hydrolase family genes and downregulation of redox-related genes with increasing bamboo proportions. Biochemical assays confirmed these findings, showing decreased oxidative substances and increased reductive compounds in mycelia grown with high bamboo content, which indicate disrupted cellular redox homeostasis. This study provides both a practical solution to alleviate the “edible mushrooms derived from lignicolous fungi–forest conflict” and fundamental insights into fungal adaptation mechanisms to non-wood substrates, thus establishing a theoretical foundation for the valorization of agricultural and forestry wastes.