Brewer’s spent grain (BSG) is the main insoluble solid by-product of the brewing industry. To add value to this non-wood material, microcrystalline cellulose (MCC) was prepared from BSG by alkaline pretreatment, bleaching, and subsequent acid hydrolysis to produce non-wood MCC. This study aimed to optimize MCC production using a statistical design (Box-Behnken) with three factors at three levels: acid concentration (0.5–1.5 M), hydrolysis time (70–90 min) and hydrolysis temperature (55–65 °C), to achieve the maximum yield and crystallinity of MCC derived from BSG. Results from 17 experimental runs revealed that the hydrolysis condition of 0.63 M HCl for 70 min at 61 °C yielded the highest output of 15% with a crystallinity index of 60%. The chemical structures and characteristics of MCC derived from BSG were verified by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction analysis (XRD). FT-IR analysis showed that the major wavenumbers of lignin and hemicellulose after the chemical processes (1500 cm–1 and 1735 cm–1) decreased by 21.40% and 4.60%, respectively. The XRD chromatogram revealed that the XRD characteristic peaks were sharper after the chemical treatments, indicating an increase in cellulose crystallinity due to removing lignin and hemicellulose. The crystallinity index of MCC derived from BSG ranged from 55–64%, which is comparable in quality to commercial pharmaceutical MCC, Avicel® PH-101 (67.37%). These results demonstrated that MCC from BSG was successfully prepared by acid hydrolysis under optimized conditions. BSG proved a viable non-wood source for preparing MCC for application in the pharmaceutical and nutraceutical industries.