Electrochemiluminescence (ECL)-functionalized metal-organic frameworks (MOFs) have attracted increasing attention in biosensing in virtue of their diverse and tunable optical properties. A famous ECL luminophore, carboxyl-rich tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) (Ru(dcbpy)32+), possesses the characteristics of good water solubility and excellent ECL performance and also has the potential to be the organic ligand of metal-organic frameworks. Herein, functionalized MOF nanosheets (RuMOFNSs) containing plenty of Ru(dcbpy)32+ in the frameworks were synthesized in aqueous solution by a simple one-pot method. In this protocol, Ru(dcbpy)32+ acted as organic ligand to coordinate with Zn2+ originated from Zn(NO3)2, and polyvinylpyrrolidone (PVP) was used as structure-directing agent to control the formation of sheetlike structure. For practical application, a "signal-on" ECL immunosensor was designed for cardiac troponin I (cTnI) detection by employing RuMOFNSs as ECL probe. The immunosensor exhibited high sensitivity and excellent selectivity for cTnI detection in the range from 1 fg/mL to 10 ng/mL with a detection limit as low as 0.48 fg/mL. Finally, the biosensor was successfully applied for the detection of cTnI in human serum sample with satisfactory results, demonstrating its potential application in bioanalysis and clinical diagnosis.