The electrochemiluminescence (ECL) behavior of N-(aminobutyl)-N-(ethylisoluminol)/hemin dual-functionalized graphene hybrids (A-H-GNs) and luminol-functionalized silver/graphene oxide composite (luminol-AgNPs-GO) was investigated under cyclic voltammetry and pulse potential. It was found that A-H-GNs and luminol-AgNPs-GO exhibited excellent ECL activity. On this basis, a label-free ECL aptasensor for 2,4,6-trinitrotoluene (TNT) detection was developed based on bilayer structure of luminescence functionalized graphene hybrids consisting of A-H-GNs and luminol-AgNPs-GO. First, positively charged chitosan-coated A-H-GNs were modified on the surface of indium-doped tin oxide electrode by simple dripping and drying in the air; after that, the modified electrode was immersed in negatively charged luminol-AgNPs-GO modified with aptamer (apta-biotin-SA-luminol-AgNPs-GO) to form apta-biotin-SA-luminol-AgNPs-GO/CS-A-H-GNs/ITO electrode (i.e., aptasensor) by electrostatic interaction. In the presence of TNT, a remarkable decrease in ECL signals was observed due to the formation of aptamer-TNT complex. TNT could be detected based on the inhibition effect. The aptasensor exhibits a wide dynamic range from 1.0 × 10(-12) to 1.0 × 10(-9) g/mL, with a low detection limit of 6.3 × 10(-13) g/mL for the determination of TNT, which is superior to most previously reported bioassays for TNT. Moreover, the proposed aptasensor has been successfully applied to the detection of TNT in environmental water. It is sensitive, selective, and simple, avoiding complicated labeling and purification procedures. Due to the wide target recognition range of aptamer, this strategy provides a promising way to develop new aptasensor for other analytes.