A β‐glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G‐200, CM‐Biogel A and DEAE‐Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a Mr of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β‐glucosidase is inhibited by gluconolactone but not by 10 mM glucose.