A novel method, based on the electrochemical manipulation of the sol-gel process, was developed to immobilize bacteria in thin hybrid sol-gel films. This enabled the safe immobilization of Escherichia coli on electrode surfaces. E. coli strains C600, MG1655 pUCD607 and MG1655 pZNTA-GFP were incorporated and physically encapsulated in a hybrid sol-gel matrix and the metabolic activity and membrane integrity of the bacteria were assessed as a function of the aging time in the absence of nutrients at +4 °C or -80 °C. LIVE/DEAD BacLight bacterial viability analysis detected by epi-fluorescence microscopy indicated the preservation of 95% of E. coli C600 membrane integrity in the sol-gel film. The presence of chitosan, trehalose and polyethylene glycol additives was shown to strongly improve the viability of E. coli cells in the electrodeposited matrix for 1 month after encapsulation. Finally, the bioluminescent activity of E. coli MG1655 pUCD607 was preserved by approximately 50% of the cells present in such composite films.