Polypyrrole (PPy) and PPy-containing chitosan-coated Fe3O4 have been electrochemically polymerized on pencil graphite electrodes (PGEs). After the resulting electrodes were characterized by SEM-EDS analysis, glucose-6-phosphate dehydrogenase (G6PD) was immobilized onto these electrodes via glutaraldehyde. The biosensors prepared for the chronopotentiometric detection of glucose-6-phosphate (G6P) at 0.25 mAcm(-2) were studied and optimized at different parameters such as the pH of supporting electrolyte, the temperature, and NADP(+) and G6P concentrations related with the analytical performance of the biosensors. PPy/G6PD (BS-1) and CS/Fe3O4-PPy/G6PD (BS-2) biosensors showed a broad linear response in the concentration range 0.025-0.25mM and 0.0025-0.05mM, and their detection limits for G6P and the RSD values were determined as 0.008mM and 0.002mM and 3.80% and 4.60% after 15 times usage, respectively. The interference study with various major blood components such as urea, glucose, and cysteine was performed to evaluate the selectivity of the biosensors. The proposed BS-2 biosensor showed almost free response from available interferences in blood serum with a recovery of 91 to 110%. The developed biosensors could be used in the G6P level measurement of medical samples.