An original electrodeposited polymer film, based on 3-ethynylthiophene, for covalent immobilization of proteins via "click" reaction of copper catalyzed azidealkyne cycloaddition was developed. The best characteristics have demonstrated the layer-by-layer immobilized 3,4-ethylendioxythiophene-3-ethynylthiophene composite film. The bovine serum albumin and antibodies have been successfully immobilized on the surface of planar plati-num electrode. The approach has been applied to labelfree electrochemical immunosensors for Escherichia coli and Staphylococcus aureus determination. The immunosensors demonstrated LODs 7.2 CFU/mL for Escherichia coli and 15.9 CFU/mL for Staphylococcus aureus, linear range 10 2 -10 6 CFU/mL and could be promising for environmental and food control.