Ratiometric electrochemiluminescence (ECL) has attracted special focus in the biological analysis field, because it could eliminate the environmental interference and allow for precise measurement. Herein, a dual-wavelength ratiometric ECL biosensor was designed for the detection of cardiac troponin I (cTnI), where (4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) (Ru(dcbpy)32+) and Au nanoparticle-loaded graphene oxide/polyethylenimine (GPRu-Au) nanomaterial acts as an acceptor, and Au nanoparticle-modified graphitic phase carbon nitride nanosheet composite (Au-CNN) acts as donor. Au-CNN shows a high and steady ECL signal centered at 455 nm, which is well-matched with the adsorption of GPRu-Au; thereby, a highly efficient electrochemiluminescent resonance energy transfer (ECL-RET) sensing platform is designed. AuNPs facilitate the immobilization of antibody on the nanomaterials through a Au-N bond. The high surface area of graphene oxide/polyethylenimine allows a large number of Ru(dcbpy)32+ to be loaded, immensely amplifying the ECL signal. This sensing platform exhibits outstanding analytical performance toward cTnI with a detection limit of 3.94 fg/mL (S/N = 3). The high reliability, selectivity, and sensitivity of this ratiometric ECL biosensor provides a versatile sensing platform for the bioanalysis.