β-D-Galactosidase from the mature tissues of green onion was purified to homogeneity using a procedure involving gel-filtration, anion-exchange chromatography, hydroxyapatite chromatography, and affinity chromatography on p-aminobenzyl-l-thio-β-D-galactopyranoside-agarose. Its molecular weight was estimated to be 52,000 Da and its isoelectric point was 6.5. The overall yield was 0.2% and specific activity was increased approximately 380 folds. Activity was maximum at pH 4 and 50℃. The purified enzyme was demonstrated to be specific to Gal-β1-6 linkage using fluorescence-labeled substrates.