Xylo-oligosaccharides (XOs) have been reported as functional ingredients due to their positive impact on human health. The XOs can be produced from lignocellulosic biomass; however, there is a lack in the literature regarding the most suitable process to recovery XOs. Due to the abundance and low cost of the lignocellulose contained in brewer’s spent grains (BSG), the main solid waste generated from beer production, this feedstock can be used in various bioprocess applications after an appropriate pretreatment, including the recovery XOs. Therefore, this study evaluated the production of XOs from BSG using a laboratory-scale flow-through subcritical water hydrolysis reactor. The hydrolysis was performed with 10 g of BSG in the reactor, for 60 min at 15 MPa, water flow of 5 mL/min, and 80, 130, and 180 °C. The results obtained demonstrated that the highest amount of total XOs could be obtained at 180 °C (981 mg/L hydrolysate). The majoritarian XOs obtained from subcritical water hydrolysis of BSG were xylotriose (391 mg/L) and xylobiose (364 mg/L). Beyond, xylotetrose (160 mg/L) and xylopentose (56 mg/L) were obtained in lower proportions. The advantage of producing high xylotriose and xylobiose is associated with a possibility to promote better prebiotic effects when compared with longer chains XOs. The XOs obtained are suitable for prebiotic applications, presenting high antioxidant activity by DPPH (0.58 mg/mL) and FRAP (0.72 mg/mL) assays. In conclusion, subcritical water hydrolysis can be a promising technology to recovery XOs from BSG.