Soybean contains anti-nutritional factor raffinose oligosaccharide (RFOs), which can cause flatulence, gastrointestinal dysfunction and low feed utilization rate. However, α-galactosidase can hydrolyze raffinose oligosaccharides (RFOs). Therefore, in this study, a novel α-galactosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 (TstGal) which can hydrolyze raffinose was cloned, expressed, purified, and characterized. The gene fragment size is 2208 bp, and the enzyme TstGal consists of 736 amino acids. Under the optimum culture conditions, the maximum enzyme activity of the target protein TstGal was 23.8 U/mL. The enzyme was purified 11.3-fold by Ni-NTA agarose resin with an overall recovery of 51.4 % and specific activity of 9.0 U/mg, and its relative molecular weight was about 85 kDa. The optimal temperature of TstGal was 70 °C, and it exhibited excellent thermal stability at 60 °C. Furthermore, The TstGal had the highest activity at pH 6.5 and good pH stability at pH 5.0-7.5. Besides, the enzyme has a good sugar tolerance to galactose and sucrose. In addition, K