It remains a great challenge to develop effective strategies for improving the weak cathodic electrogenerated chemiluminescence (ECL) of the luminol-dissolved O2 system. Interface modulation between metal and supports is an attractive strategy to improve oxygen reduction reaction (ORR) activity. Therefore, the design of electrocatalysts via interface modulation would provide new opportunities for the ECL amplification involving reactive oxygen species (ROSs). Herein, we have fabricated an Ag single-atom catalyst with an oxygen-bridged interface (Ag-O-Co) through the electrodeposition of Ag on a CoAl layered double hydroxide (LDH) modified indium tin oxide (ITO) electrode (Ags/LDH/ITO). Interestingly, it was found that the cathodic ECL intensity of the luminol-dissolved O2 system at the Ags/LDH/ITO electrode was extraordinarily enhanced in comparison with those at bare ITO and other Ag nanoparticle-based electrodes. The enhanced ECL performances of the Ags/LDH/ITO electrode were attributed to the increasing amounts of ROSs by electrocatalytic ORR in the Ag-O-Co interface. The electron redistribution of Ag and Co bimetallic sites could accelerate electron transfer, promote the adsorption of O2, and sufficiently activate O2 through a four-electron reaction pathway. Finally, the luminol cathodic ECL intensity was greatly improved. Our findings can provide inspiration for revealing the interface effects between metal and supports, and open up a new avenue to improve the luminol cathodic ECL.