Abstract The rutin-hydrolyzing enzyme (RHEs) catalyse hydrolysis of rutin to quercetin. In this study, a RHE purified from natural tartary buckwheat flour (FtRHE) exhibited β-glucosidase activity. It was highly active on cello-oligosaccharides and p-nitrophenyl-β-D-glucopyranoside (pNPG). FtRHE exhibited optimum β-glucosidase activity at 40 °C and pH 4.0, which was elevated by the presence of Ca2+, suggesting that Ca2+ may act as a co-factor for its activation. The Km and Vmax of FtRHE were 0.22 mM and 310.48 U/mg, respectively, when pNPG was the substrate. FtRHE retained only 6.6% activity in the presence of 1.5 M glucose, indicating that glucose acted as its inhibitor. This study demonstrates β-glucosidase activity of a RHE from natural tartary buckwheat flour and provides better understanding of its role in glycoside metabolism, establishing a basis for further investigations.