In this work, Pt/Au bimetallic nanoparticles (Pt/Au NPs) were used as nanocarriers to develop an electrochemiluminescence (ECL) immunosensor for sensitive cardiac troponin I (cTnI) detection, coupling with enzyme-based signal amplification. First, gold nanoparticles modified Ru(phen)3(2+)-doped silica nanoparticles (Au@RuSiO2 NPs) with numerous luminophores were used as a platform, potentially increasing the signal intensity. Second, Pt/Au NPs with large surface area and rich surface atoms were a superior matrix for the immobilization of numerous antibodies (Ab2), poly(l-histidine) (PLH) and glucose dehydrogenase (GDH). More importantly, the PLH-protected GDH exhibited excellent enzymatic activity for the oxidation of glucose accompanied by the reduction of NAD(+) to NADH. The in situ generated NADH acted as a co-reactant of Ru(phen)3(2+), significantly enhancing the ECL signal. In this manner, the designed immunosensor displayed high sensitivity for the detection of cTnI in the range of 0.010 ng mL(-1) to 10 ng mL(-1) with a detection limit of 3.3 pg mL(-1) (S/N = 3). The proposed strategy holds a new promise for highly sensitive bioassays for application in clinical analyses.