A self-enhanced electrochemiluminescent ratiometric immunoassay for zearalenone is described. A system composed of N-aminobutyl-N-ethylisoluminol (ABEI) and glutathione (GSH) produces a strong electrochemiluminescence (ECL) at an applied potential of 0.8 V, probably because of short electron transfer distance and reduced energy loss. The method also uses octahedral anatase mesocrystals (OAM) with a large specific surface facilitating immobilization of ABEI and GSH. Helical carbon nanotubes, possessing a large specific surface, superior mechanical stability, and excellent electrical conductivity which serve as a solid support, greatly enhanced the loading capacity for g-C3N4 nanosheets and horseradish peroxidase-labeled anti-antibody. The peroxidase accelerates the decomposition of H2O2 to produce reactive oxygen species (ROSs), amplifying the blue ECL of ABEI and the green ECL of g-C3N4. The ratiometric sandwich immunoassay (performed by the ratio of ECL intensity at − 1.3 V and 0.8 V) allows for sensitive and reliable determination of ZEN in a wide linear range from 1.0 × 10−4 ng/mL to 10 ng/mL. The method was successfully applied to the analysis of corn hazelnut samples for ZEN. Graphical abstractSchematic presentation of a self-enhanced electrochemiluminescent ratiometric immunosensor based on octahedral anatase mesocrystals (OAM) supported ABEI-glutathione (GSH) and g-C3N4 functionalized helical carbon nanotubes (HCNT) for zearalenone (ZEN) determination.