Abstract A sensitive electrochemical immunosensor for 3-bromobiphenyl (3-BBP) detection was constructed by employing a new polydopamine coated prussian blue-mesoporous carbon (PDOP/PB/CMK-3) nanocomposite as the substrate platform and multi-horseradish peroxidase-double helix carbon nanotubes-secondary antibody (multi-HRP-DHCNTs-Ab2) as the signal label. PB/CMK-3 was firstly successfully in-situ synthesized with the aid of the CMK-3 reduction, which was characterized by transmission electron microscope (TEM), infrared spectroscopy (IR), X-ray diffraction (XRD) and N2 adsorption–desorption analysis. By using PDOP/PB/CMK-3 as the substrate, it can effectively enhance the specific surface for antigen loading due to the three-dimensional structure of the nanocomposites, while large amount of PB that fixed inside or outside the pore of CMK-3 successfully improved the electrochemical response and the PDOP film can provide a biocompatible environment to maintain the activity of antigen availability. Under the optimized conditions, the proposed immunosensor shows a good current response to 3-BBP in a linear range from 5pM to 2nM with a detection limit of 2.25pM. In addition, the specificity, reproducibility and stability of the immunosensor were also proved to be acceptable, indicating its potential application in environmental monitoring.