This study was conducted to evaluate the potential of using domestic agricultural by-products—pruned branches of apple and pear trees, soybean stems, and spent cocopeat substrate—as alternative substrates for the cultivation of Flammulina velutipes. Chemical analysis showed that Substrate 1 (apple branches replacing corncob) and Substrate 2 (pear branches replacing corncob) had total carbon (42.6%) and total nitrogen (1.5%) contents most comparable to those of the Control. Mycelial growth in all mixed substrates exceeded that of the control. Fruiting body yield was higher in Substrate 2 (273.4 g/1,100mL) and Substrate 1 (238.0 g) compared with the Control (231.5 g). Fruiting body quality in Substrates 1 and 2 was equivalent to that of the control. These results indicate that woody by-products such as apple and pear pruned branches can serve as effective replacements corncob in F. velutipes cultivation.