Pretreatment and detoxification processes are essential for conventional cellulase production from lignocellulose. However, spent mushroom substrate (SMS) can be potentially used for cellulase production without these processes. In this study, lignocellulose contents in various generations of SMS used for cultivating Auricularia polytricha, Auricularia nigricans, and Pleurotus ostreatus were analyzed. The highest lignin degradation ability was observed in SMS used for cultivating A. polytricha, whereas the highest cellulose degradation ability was observed in SMS used for cultivating A. nigricans. The production of cellulase using the earlier generation of SMS was higher than that using the later generation, and the highest cellulase activity from the third-generation SMS used for A. polytricha was 3.51 U/mL. The study also demonstrates that the use of ultrasonic treatment in the fermentation process could improve the cellulase activity, as was evident by the increase of cellulase activity by 24.5 % after ultrasonic treatment for 40 min at 0.8 duty cycle after 4 days of fermentation. This study not only provides a possible solution for SMS disposal, but also presents a substrate that can potentially be used for cellulase production without the need of pretreatment.