Vitexin is an active component of many traditional chinese medicines, and is found in various plants. The low solubility of vitexin limits its pharmaceutical usage. In this study, solvent-stable β-fructosidase was used to glycosylate vitexin in organic solvents. The β-fructosidase showed high activity and stability in 30–80% (v/v) ethyl acetate with 90–99% yields of vitexin glycosides. Highly efficient synthesis of β-D-fructofuranosyl-(2→6)-vitexin (1.04 g L⁻¹) and β-D-difructofuranosyl-(2→6)-vitexin (0.45 g L⁻¹) was attained in 50% (v/v) ethyl acetate solvent system from 1.5 g L⁻¹ vitexin. Two novel vitexin glycosides showed higher anti-tumor activities compared to that of vitexin by employing a human breast cancer cytotoxicity assay.