In this method, an intermediate polyelectrolyte layer is first adsorbed on an oppositely charged membrane by electrostatic interactions. This leads to a charge inversion of the original membrane. Then the bio-macromolecule is bound to the intermediate polyelectrolyte layer, always by charge interactions. The method feasibility was shown with two bio-macromolecules, glucose oxydase and heparin, which were immobilized on negatively-charged membranes via polyethyleneimine, a cationic hydrophilic polymer. The immobilization of glucose oxydase on different polymer membranes led to high-activity and stable membranes for glucose biosensor. The anti-coagulation effect of immobilized heparin was not clearly evidenced in spite of the effective bio-species immobilization. The good properties of the immobilized enzyme was explained by the hydrophilicity of the intermediate polyelectrolyte layer, its high density in sites for enzyme binding, and the mild immobilization conditions.