Electrosynthesized poly(1,6-hexanedithiol) (PHDT) was proposed as a new immobilization matrix for Au-nanoparticles-enhanced piezoelectric immunosensing, with human immunoglobulin G (hIgG)/anti-hIgG as an example. In ethanol containing 0.10M LiClO4, the electropolymerization of 1,6-hexanedithiol (HDT) occurred through the oxidation of the –SH groups of HDT and then –S–S– linking at potentials positive of ∼0.7V vs. SCE, as characterized by the electrochemical quartz crystal microbalance (EQCM). PHDT was prepared potentiostatically at 1.25V vs. SCE, and Au nanoparticles (AuNPs) were then immobilized on the prepared PHDT films by dip-dry coating. The immobilization amount of AuNPs was investigated vs. the volume of dipped AuNPs solution, and a theoretical discussion is given on the coverage of AuNPs on the PHDT film. Under the optimized experimental conditions, the adsorption amount of anti-hIgG was the greatest on the AuNPs/PHDT/Au electrode, as compared with those at the bare Au and PHDT/Au electrodes. The molar combination ratio of anti-hIgG to hIgG was estimated to be 1:0.493 on the AuNPs/PHDT/Au electrode in a subsequent immunoreaction. Compared with the common immobilization protocol with HDT, namely, AuNPs immobilized directly on an HDT self-assembly (HDT-SA) at an Au electrode (AuNPs/HDT-SA/Au), the AuNPs/PHDT/Au electrode shows better stability and can be constructed more time-effectively, though the adsorption amount of anti-hIgG was comparable with each other. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were utilized for characterizations of and comparison among various surfaces. The electrosynthesized PHDT presented here as a new bio-immobilization matrix is highly recommended for wider applications in biosensing.