On-site production of cellulases using lignocellulosic materials can improve the economic viability of biorefineries. This, however, requires the pretreatment of substrates using thermochemical conditions that can vary in severity. To understand the effect of pretreatment severity on cellulase production by Aspergillus ustus S3 on sugarcane bagasse, we applied NaOH pretreatment corresponding to 3 severity factors (SF1.32, SF1.79, and SF3.64) to generate SCB that was used as inducing substrate. The highest cellulase activity (0.681 U/mL) was obtained with the intermediate severity (SF1.79) while significantly lower activities of 0.495 and 0.539 U/mL were recorded with low (SF1.32) and high (SF3.64) severities, respectively. Chemical and structural characterization revealed that low and intermediate severities improved cellulose accessibility and cellulase titres while high severity impaired them, thus limiting substrate suitability for enzyme production. These results show that though high SFs might be desirable in other applications, moderate severities may be more appropriate for cellulase production.