A membrane was prepared using polyvinyl alcohol (PVA) with low and high degree of polymerization (DOP), acetone, benzoic acid (BA) and was cross-linked by UV treatment. Membrane composition was optimized on the basis of swelling index. Membrane prepared with 12% low DOP and 8% high DOP of PVA, 2% BA, dissolved in buffer containing 20% acetone and cross-linked with UV treatment exhibited lower swelling index. Fourier transform infrared (FTIR) study of the membranes showed appearance of a strong band at ∼2337 cm−1 when UV was used for cross-linking in the presence of benzoic acid. Scanning electron microscope (SEM) study revealed that membrane cross-linked with UV treatment was smoother. Glucose oxidase (GOD)–PVA membrane was associated with the dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose to gluconolactone. A wide detection range, 0.9–225 mg/dl was estimated from the linear range of calibration plot of biosensor reading. Membranes were reused for 32 reactions without significant loss of activity and stored for 30 days (∼90% activity) at 4 ◦C. Membranes were also used with real blood samples.