In this article, we describe the preparation of a new lactose biosensor based on electrode coating with ss-galactosidase and glucose oxidase immobilized gelatin. For this purpose, ss-galactosidase and glucose oxidase enzymes were immobilized onto gelatin by crosslinking with glutaraldehyde. Properties of the immobilized ss-galactosidase and glucose oxidase enzymes electrode have been studied. The effects of glutaraldehyde concentration, temperature and pH variations and reusability were among the subjects analyzed. Lactose biosensors were subjected to continuous repeated use in order to observe reusability and shelf life; where standard lactose and milk samples were used as substrate solutions. Continuous reuse experiments showed that most of the lactose biosensors activities were retained even after the 10th use in a period of 30 days.