Covalently modifying electrochemiluminescence (ECL) luminophores to alter their energy levels or generate energy/electron transfer processes for improved performance is hindered by the complex design and fabrication processes. In this study, non-covalent bond self-assembly was employed to enhance the ECL property of gold nanoclusters with tryptophan (Try) and mercaptopropionic acid (MPA) as ligands (Try-MPA-gold nanoclusters). Specifically, through the molecular recognition of Try by cucurbit[7]uril, some non-radiative transition channels of the charge carriers on the surface of the Try-MPA-gold nanoclusters were restricted, resulting in a significant enhancement of the ECL intensity of the nanoclusters. Furthermore, rigid macrocyclic molecules acted on the surface of the nanoclusters through self-assembly, forming a passive barrier that improved the physical stability of the nanoclusters in the water-phase and indirectly improved their luminescent stability. As an application, cucurbit[7]uril-treated Try-MPA-gold nanoclusters (cucurbit[7]uril@Try-MPA-gold nanoclusters) were used as signal probes, and Zn-doped SnO2 nanoflowers (Zn-SnO2 NFs) with high electron mobility were used as electrode modification material to establish an ECL sensor for kanamycin (KANA) detection, utilizing split aptamers as capture probes. The advanced split aptamer sensor demonstrated excellent sensitivity analysis for KANA in complex food substrates with a recovery rate of 96.2 to 106.0%.