Glucose-isomerase activity was optimized on ripened jackfruit pulp (Artocarpus heterophyllus). The GI action on 20% pulp extract of ripe jackfruit was investigated after it was analyzed for fructose content, reducing sugar, non-reducing sugar and total sugar which were found 7.53 g/100g, 11.56 g/100g, 5.10 g/100g and 16.93g/100g respectively. The GI activity was found bi-directional at pH 7.0, temperature 70°C, time 120 min on 5 ml substrate with 100 mg of GI concentration. The negative value (-0.08 µmoles/min) of GI activity on 20% pulp extract indicated the reverse reaction and was found dependent on the concentration of glucose and fructose present in the jackfruit pulp which further confirmed by enriching the pulp with 10% glucose and 10% fructose. Due to the reverse direction of GI activity on jackfruit pulp, the fructose was separated from the pulp before the application of GI, leaving glucose for isomerization to get high fructose sugar. Keywords: Jackfruit; Glucose-isomerase; Glucose; Fructose; High fructose sugar.