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The heavy reliance of the mushroom industry on sawdust substrates is putting increasing pressure on already limited forest resources, forcing researchers to seek alternative materials. This study investigated the feasibility of using post-harvest djulis (Chenopodium formosanum Koidz.) stems, waste from this indigenous crop in Taiwan, to partially replace sawdust for Pleurotus sajor-caju cultivation. Initial screening with 0–100% djulis replacement revealed growth inhibition above 50% incorporation levels. Refined experiments focusing on 0–30% djulis ratios demonstrated that strain PT exhibited superior adaptation to djulis-containing substrates. Commercial scale grow bag trials showed that among djulis treatments, 25% djulis incorporation achieved the fastest mycelial colonization rate (1.0 cm/day), while 15% incorporation yielded the highest biological efficiency (76.17%), comparable to commercial controls (76.80%). Three-flush harvest cycles confirmed stable productivity across treatments, with total yields ranging from 286 to 320 g/bag. Nutritional analysis showed no major changes in amino acids and antioxidants, with djulis incorporation maintaining protein quality while some enhancement in total free amino acid content and reducing power at 25% incorporation. These findings demonstrate that 15–25% djulis stem substitution sustained commercial production parameters while contributing to sustainable agricultural waste management and reducing forest resource dependence.