In this paper, an egg-shell membrane has been used for efficient immobilization and stabilization of glucose oxidase. This membrane was used for developing a simple and reusable method for estimation of glucose in biological samples. The proposed sensor was effectively used in a wide glucose concentration range (1 - 1000 mM) with fast response time of 70 s for higher concentrations and 120 s for lower concentrations. The results of response study for the fabricated sensor show limit of detection of 4.761 mM with high sensitivity over the entire concentration range (1 - 1000 mM). Most interestingly, the membrane used in the fabricated sensor could be repeatedly used for glucose analysis 150 times and it exhibited a shelf-life of more than 6 weeks. The proposed sensor was also demonstrated for estimation of glucose in human blood samples.