A biosensor was fabricated with glucose oxidase (GOD) immobilized on self-assembled monolayers (SAMs)/Prussian blue (PB)/chitosan (Chit). The electrochemical characteristics of the biosensor were studied in the presence of dipolar aprotic organic solvents such as acetonitrile (AN), dimethyl sulfoxide (DMSO) and N,N-dimethyl formamide (DMF). The effect of volume percentage of organic solvents on biosensor response was investigated. It is observed that the response current of the biosensor in 50% AN reaches 73% of that in phosphate buffer solution (PBS). The amperometric signals are fast, reproducible and exhibiting high sensitivity (57.3mA·M·cm-2). The biosensor was used to determine glucose in serum specimens, and the results are consistent with those obtained with the commercially available glucose enzyme photometric kit.