A novel approach for the in situ synthesis of conjugated polyaniline-poly(ethylene glycol) graft copolymer (PA-g-PEG) by the combination of oxidative polymerization and copper catalyzed azide alkyne cycloaddition (CuAAC) click reaction is described. The method pertains to the reduction of the CuBr2 catalyst during the oxidative copolymerization of aniline and aminophenyl propargylether to Cu(I) species, which catalyze the CuAAC reaction between thus formed polyaniline with pendant alkyne groups and independently prepared azide functional PEG in both simultaneous and sequential manner. The obtained water-soluble (PA-g-PEG) was used for the construction of glucose biosensor by a simple one-step approach. Combined electrostatic polyanion-polycation and hydrogen bond interactions between (PA-g-PEG) and glucose oxidase provided a suitable immobilization matrix for the enzyme resulting in excellent analytical parameters. (PA-g-PEG) based glucose biosensor exhibited a remarkable response time, producing an instant signal upon addition of analyte, making this sensor an attractive alternative for the existing devices.