To promote the comprehensive utilization of Pueraria lobata (P. lobata) straw, this study explored the antioxidant activity and probiotic activity of its xylooligosaccharides (XOS). The hemicellulose B from P. lobata straw was extracted using alkali solution. The protein was removed by Sevag method and analyzed by ultraviolet-visible full wavelength scanning. The monosaccharide composition of hemicellulose B was determined using high performance liquid chromatography (HPLC) after acid hydrolysis. XOS were obtained after xylanase hydrolysis. XOS were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR). Subsequently, the antioxidant activity and probiotic activity of XOS were verified. Full wavelength scanning revealed the successful removal of protein had been removed by Sevag method. HPLC analysis indicated that the hemicellulose B of P. lobata straw contained L-guluronic acid, D-mannose, D-glucosamine, L-rhamnose, D-glucuronic acid, D-galactosamine, D-glucose, D-galactose, D-xylose, L-arabinose, and L-fucose. Among them, the contents of xylose and glucose were relatively high, accounting for 37.831% and 23.179%, respectively. SEM observation showed that XOS were fragmented and had a rough surface. FT-IR analysis revealed that XOS mainly contain beta-glucosidic and alpha-glucosidic bonds. NMR analysis showed that XOS contain methoxy group. The activity experiment demonstrated that XOS exhibit strong antioxidant activity and reducibility, and are beneficial to Lactobacillus acidophilus and Bifidobacterium animalis. Intestinal simulation experiment demonstrated that XOS have an inhibitory effect on Escherichia coli.