To achieve a high efficiency of analyte capture by a capture antibody attached to an electrochemical immunosensor, we have immobilised an analyte-specific antibody on a self-assembled layer of recombinant Protein G that was thiolated with succinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP). Then two techniques were employed for conjugating a second antigen-specific antibody to alkaline phosphatase (mAb2-AP) using either LC-SPDP or the biotin–streptavidin interaction as the mode of cross-linking the antibody and enzyme. After characterising the two mAb2-AP preparations (mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP), they were each used as the signal antibody for immunosensors formatted for two-site immunoassays where the capture antibody was attached to a Protein G-(LC-SPDP) scaffold on gold electrodes. The antibodies and assays were specific for the clinically important hormone, human chorionic gonadotrophin (hCG). Protein G-(LC-SPDP) provided a stable scaffold, while mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP performed well as the signal antibodies. Immunosensors with mAb2-(Biotin-SA)-AP were characterised by a limit of detection of 216 IU L⁻¹ for hCG and a linear response up to approximately 2000 IU L⁻¹. Conversely, immunosensors with mAb2-(LC-SPDP)-AP exhibited a limit of detection of 240 IU L⁻¹ and a linear response up to 4000 IU L⁻¹.