An α-l-rhamnosidase secreted by Penicillium citrinum MTCC-8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation-exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The Km and Vmax values of the enzyme for p-nitrophenyl α-l-rhamnopyranoside were 0.36 mm and 22.54 μmole min−1 mg−1, respectively, and kcat value was 17.1 s−1 giving kcat/Km value of 4.75 × 104 m−1 s−1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l-rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l-rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α-l-rhamnose and also in the enhancement of wine aroma.