The present study deal with hydrolysis of lactose by kluyveromyces fragilis that grown in Whey and synthetic mediumandthe effect of incubated temperature and pH on enzyme activity Lactase (ß- galactosidase) that hydrolyze lactose in whey milk and synthetic medium .Also the conversion of glucose that released from lactose hydrolysis by kluyveromyces fragilis into fructose via addition of glucose isomerase. The present work showed that the optimum pH and incubated temperature were (4,35°C) respectively to optimum enzyme activity Lactase with total glucose reached 60.21 mg/ 100ml in whey medium and 58.61 mg/100ml in synthetic medium after 24 hours. In addition fructose content was 48.15 mg/100ml from released glucose in whey milk that confirmed due to the highest sweet syrup. In conclusion, this study indicated that ability of kluyveromyces fragilis to hydrolyze lactose in natural whey milk higher than synthetic medium.