A novel biosensor based on the Sodium alginate (SA), Polyvinyl alcohol (PVA) and Polyethylene glycol (PEG) was fabricated for the determination of oxygen, hydrogen peroxide (H2O2) and nitrite. The stable composite SA-PVA-PEG/Mb blend microspheres iflm were characterized by scanning electron microscopy (SEM), UV–vis spectroscopy and electrochemistry. The practical feasibility of the sensor has been addressed.