Bananas are the most important fruit crop in the world. Short shelf life of the fruit is the major limiting factor in international trade, and this is due to the softening of the pulp during ripening. Fruit softening is an important aspect of ripening process in fleshy fruits and is caused by the cumulative action of a group of cell wall‐modifying enzymes. Polygalacturonase (PG) is the key enzyme involved in the fruit softening process in banana, and this study reports the isolation, purification and characterisation of polygalacturonase enzyme from ripened fruits of a delayed ripened banana cultivar found specifically in Kerala (Musa acuminata cv. Kadali). PG was purified by ammonium sulphate fractionation followed by DEAE cellulose ion exchange chromatography and gel filtration using Sephadex G 100. The purified protein showed two subunits on SDS‐PAGE and a single band on native PAGE. Enzyme showed maximum activity at pH 3.5 and 40 °C. Fe3+ enhanced the activity more, while Mg2+ and Ca2+ slightly stimulated the activity of purified enzyme. Km value for substrate polygalacturonic acid was 0.06%.