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<i>Pleurotus ostreatus</i> (Jacq.) P. Kumm is the second most cultivated edible mushroom worldwide. Temperature is one of the main environmental factors affecting the growth and fructification of mushrooms. However, the effects of ambient temperature on the growth, development, and production of bag-cultured <i>P. ostreatus</i> are still unclear. In this study, eight ambient temperatures (15 °C, 18 °C, 20 °C, 22 °C, 25 °C, 28 °C, 30 °C, and 32 °C) were used to cultivate <i>P. ostreatus</i> mycelia with cottonseed hull as substrate. Then, the agronomic traits and the activities of substrate-degrading and antioxidant enzymes were determined. The mycelial growth and fructification of <i>P. ostreatus</i> differed significantly under different temperatures, and 22 °C was the optimum temperature for mycelial culture when we comprehensively analyzed all the agronomic traits. The mycelial growth rate and the biological efficiency under 22 °C were 0.56 cm/d and 99.53%, respectively. Furthermore, there were also significant differences in the activities of substrate-degrading enzymes and antioxidant enzymes under different ambient temperatures. The highest activities of lignocellulose-degrading enzymes (laccase, xylanase, and cellulase), amylase, and proteinase at the vegetative growth stage occurred at 15–22 °C, 20–22 °C, and 22–30 °C, respectively. The highest activities of superoxide dismutase, peroxidase, and catalase at the vegetative growth stage occurred at 28–30 °C. This study provides a reference temperature for bag-cultured <i>P. ostreatus</i>.