Potential-resolved electrochemiluminescence (PRECL)-based strategies utilizing novel nanoluminophores have become a research hotspot in analytical sciences in virtue of the superior versatility, specificity, and sensitivity they offer to bioassays. In this work, novel PRECL nanoluminophore graphitic carbon nitrides (g-C3N4) functionalized by N-(aminobutyl)-N-(ethylisoluminol) (ABEI) (g-C3N4/ABEI) were developed, exhibiting two potential-resolved ECL emissions in the presence of H2O2/K2S2O8 as coreactants with the peak potentials at +1.2 and -1.6 V, respectively. It was found that Hg2+ could simultaneously decrease the ABEI electrochemiluminescence (ECL) intensity while increasing the intensity of g-C3N4 on the contrary. Using Hg2+ as the ECL signal moderator based on specific interactions between Hg2+ and thymine-thymine pairs, conjoined with the synthesized g-C3N4/ABEI/Hg2+ nanohybrids with outstanding PRECL properties as the sensing interface, a label-free and all-in-one ratiometric ECL bioassay combined with a rolling circle amplification (RCA) strategy was designed for the detection of miR-133a as a biomarker in early diagnosis of acute myocardial infarction (AMI). The proposed bioassay provided a quantitative readout proportional to the target miR-133a concentrations in the range from 0.1 fM to 1.0 pM with a detection limit of 48.0 aM. Owing to its inherent merits for an effective amplification of the ECL signals ratio and a simple one-step assembly procedure, the proposed bioassay demonstrated excellent analytical performance with remarkable sensitivity, specificity, and low measurement deviations, manifesting its potential application in early AMI diagnosis.