The authors describe a nanocomposite prepared from gold nanoparticles, branched polyethylenimine and carboxylated multiwalled carbon nanotubes (AuNP/b-PEI/c-MWCNTs), and its application to a highly sensitive electrochemical immunosensor for the soluble CD40 ligand (sCD40L), a biomarker of acute coronary syndrome (ACS). It is known that c-MWCNTs possess a remarkably good electrochemical conductivity that can improve the sensitivity of an immunosensor, but their tendency towards aggregation is an obstacle. The use of b-PEI can substantially decrease the tendency towards aggregation. AuNPs with their large specific surface and good electrical conductivity were used to load the antibody. The nanocomposite was characterized by transmission electron microscopy, atomic force microscopy, energy dispersive spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. When using hexacyanoferrate as an electrochemical probe (best detected at 0.18 V vs. SCE), the electrode displays a linear response in the 10 fg mL−1 to 100 pg mL−1 sCD40L concentration range, and a lower detection limit of 3 fg mL−1. The electrode is regenerable. It was applied to the determination of sCD40L in spiked serum samples, and the results agreed well with an ELISA.