Abstract: β-galactosidase was partially purified 2.16-fold with a total yield of 21.32% of the original activity by sequential use of ammonium sulfate precipitation and gel filtration through Sephadex G-100 from Durian fruit. A progressive increase in activity of the purified enzyme was observed up to 60oC accompanied by a decrease thereafter and the enzyme activity was linear with time at least up to 10 min reaction time, the maximum activity reached it after 20 min and still constant thereafter. An energy of activation of 3.04 Kcal/mole for the enzyme activity was derived from the Arrhenius plot. The optimum pH was 3.0. The purified enzyme started loosing activity above 40 oC when heated for 10 min. and became completely inactive at 80oC. Michaelis-constant of (Km) values of 5.5mM and a maximum velocity (Vmax) of 0.9 μmoles/mg/min. A Molecular weight (MW) determination of ~122 kDa was estimated by gel filtration methods using a Sephadex G-100. Fe+++, Zn++ and Cu++ strongly inhibited the enzyme. However, Mg++ , Ca++ and Mn++ inhibited negligibly.