Effective assay of Escherichia colt (E. colt) as indicator bacteria is critical for evaluating the recycling of urban sludge. In this work, we utilized multiwalled carbon nanotube/gold nanoparticle (CNT/AuNP) hybrid nanostructures as sensing electrode to design a novel electrochemical immunosensor for E. colt analysis. Herein, the hybrid nanostructures were fabricated by electrodepositing AuNPs on CNTs surface via multi-potential step technique. The CNT/AuNP hybrids were proved to facilitate the improvement of electrochemical performance for signal amplification, as well as the immobilization of E. colt capture antibody ((c)Ab). The sandwich-type system was formed by specific recognition of the immobilized cAb on CNT/AuNP hybrids to E. coli, followed by the attachment of horseradish peroxidase-conjugated E. colt detection antibody ((d)Ab-HRP) to produce the electrochemical immunosensor. On the basis of the dual signal amplification of CNT/AuNP hybrid nanostructures and enzymatic catalysis, the electrochemical immunosensor exhibits an excellent performance for E. colt assay ranging from 2.0 x 10(2) to 2.0 x 10(6) cfu mL(-1). The electrochemical method was also successfully used to evaluate E. colt in urban sludge. The proposed strategy provides a convenient and effective method for E. coli detection, and could become a promising technique for estimating the feasibility of sludge recycling. (C) 2014 Elsevier Ltd. All rights reserved.