A sensitive electrogenerated chemiluminescence peptide-based (ECL-PB) biosensing method for the determination of protein was developed by employing peptide-integrating Ru(bpy)₃²⁺(bpy = 2,2′-bipyridine)-functionalized gold nanoparticles (Ru(bpy)₃²⁺-AuNPs-peptide) as nanoprobe. Cardiac troponin I (cTnI), a reliable clinical biomarker for the detection of cardiac injury, was chosen as target protein, while a specific binding peptide (CFYSHSFHENWPS) was used as molecular recognition element. AuNPs were firstly functionalized with Ru(bpy)₃²⁺ through electrostatic interactions between citrate-capped AuNPs and Ru(bpy)₃²⁺ to form Ru(bpy)₃²⁺-AuNPs aggregates and then functionalized with peptide through Au-S bounds to form Ru(bpy)₃²⁺-AuNPs-peptide nanoprobe. AuNPs not only can capture numerous signal-generating molecules, resulting in high ECL intensity but also can capture a significant amount of the peptide, providing poly binding motif. The specific capture peptide was self-assembled on the surface of a gold electrode and then incubated with the target cTnI and Ru(bpy)₃²⁺-AuNPs-peptide successively. A sandwich-type peptide/cTnI/Ru(bpy)₃²⁺-AuNPs-peptide conjugate was formed on the surface of the electrode and an ECL signal was obtained in the presence of tri-n-propylamine. The novel biosensing method facilitates the sensitive detection of cTnI in the range from 3.0 × 10⁻¹² g mL⁻¹ to 7.0 × 10⁻¹¹ g mL⁻¹ with a low detection limit of 0.5 pg mL⁻¹. This work provides a promising strategy for the determination of proteins with simplicity, high sensitivity, and selectivity.