Here, we have reported one step fabrication of a porous thin film of the zinc oxide nanoparticles (ZnO NPs) directly onto the indium tin oxide (ITO) glass substrate by cathodic electrodeposition from an aqueous solution containing zinc nitrate. The morphological studies of a thin film of ZnO NPs suggest that the NPs formed a porous structure with irregular pore size distribution over the surface of ITO glass substrate. The average pore size and wall thickness of pores obtained as 620 nm and 50-120 nm, respectively. Transmission electron microscopic investigations reveal the growth of spherical ZnO NPs of size ranging from 20-30 nm. The surface charge on the electrodeposited ZnO NPs has shown a positive value of 18 mV as confirmed by zeta potential. This electrodeposited porous thin film of ZnO NPs has been employed for immobilization of monoclonal antibodies (Ab) specific to Vibrio cholerae for detection of Vibrio cholerae toxin B. Electrochemical studies reveal that these ZnO/ITO electrodes provide favorable conditions for immobilization of Ab with better orientation, resulting in enhanced electron transfer at the interface. This immunoelectrode exhibits improved biosensing characteristics as detection range varies from 12.5-500 ng mL(-1), the sensitivity of 71 nA ng(-1) mL cm(-2), the low detection limit of 0.16 ng mL(-1) and an association constant of 14.12 ng mL(-1). Significantly, the electrodeposited ZnO thin films are uniform, cost-effective, reproducible and stable. This can be processed for commercialization. (C) 2016 The Electrochemical Society. All rights reserved.