A new method for the formation of electroactive polyaniline(PANI) biocatalyzed by hemoglobin coupled with glucose oxidase in neutral medium on the polystyrene nanospheres(PS) modifed glassy carbon electrode, was investigated. The bio-polymerized PANI formed on the PS was confrmed by the obvious increase of the diameter of the particles on the scanning electron microscopy image. The cyclic voltammetric behavior of the PANI was also investigated. PANI produced an oxidative peak at 0.28 V and a reductive peak at 0.23 V. Based on the glucose-dependent bio-polymerization, a new electrochemical protocol for the estimation of glucose was developed. The square wave voltammetric response of PANI deposited on the modifed electrode increased linearly with glucose concentration in the range of 0.1–10.0 μmol/L. The effcient performance of hemoglobin-oxidase biocatalyzed polymerization of aniline provides a new concept for the synthesis of nanomaterials, and a general protocol for the development of the biosensors. A new method for the formation of electroactive polyaniline (PANI) biocatalyzed by hemoglobin coupled with glucose oxidase in neutral medium on the polystyrene nanospheres (PS) modifed glassy carbon electrode, was investigated. The bio-polymerized PANI formed on the PS was confrmed by the obvious increase of the diameter of the particles on the scanning electron microscopy image. The cyclic voltammetric behavior of the PANI was also investigated. PANI produced an oxidative peak at 0.28 V and a reductive peak at 0.23 V. Based on the glucose-dependent bio- The square wave voltammetric response of PANI deposited on the modifed electrode increased linearly with glucose concentration in the range of 0.1-10.0 μmol / L. The effcient performance of hemoglobin-oxidase biocatalyzed polymerization of aniline provides a new concept for the synthesis of nanomaterials, and a general protocol for the development of the biosensors