In this study, a functionalized nanocomposite-based electrochemiluminescence (ECL) sensor for detecting thrombin was developed. First, Ru(bpy)32+/β-cyclodextrin-Au nanoparticles (β-CD-AuNPs)/nanographene (NGP) composites were used to modify the glassy carbon electrode (GCE) surface, and then aptamers (TBA1 and TBA2 with a 1:1 M ratio) were labeled with ferrocene (Fc) acting as the probes and were attached to the composite via the host–guest recognition between β-CD and Fc. In the absence of thrombin, the quenching of Fc to [Ru(bpy)3]2+ was maintained, and “signal-off” ECL was observed. However, because of the specific combination of the aptamer probes and thrombin, the configuration of aptamer probes changed and escaped from the electrode surface once thrombin appears, which results in the quenching disappearance, and the ECL signal was changed from “off” to “on.” Meanwhile, the application of nanocomposites amplified the effect of “signal-on.” By this strategy, thrombin was detected with high sensitivity and with a detection limit down to 0.23 pM. Moreover, the relatively simple ECL sensor exhibited excellent reproducibility with at least 6 cycles of recovering the original signal.