In view of the outstanding catalytic efficiency, single-atom catalysts (SACs) have shown great promise for the construction of sensitive chemiluminescent (CL) platforms. However, the low loading amount of active sites dramatically obstructs the improved catalytic activity of these metal SACs. Benefiting from the exceedingly unique catalytic properties of the metal–metal bonds, atomic clusters may give rise to enhancing the catalytic properties of SACs based on the synergistic effects of dual atomic-scale sites. Inspired by this, atomic Co3N clusters-assisted Co SACs (Co3N@Co SACs) were synthesized through a facile doping method. Through X-ray absorption spectroscopy, the active metal sites in the synergetic dual-site atomic catalysts of Co3N@Co SACs were confirmed to be Co–O4 and Co3–N moieties. Co3N@Co SACs served as a superior co-reactant to remarkably enhance the luminol CL signal by 2155.0 times, which was prominently superior to the boosting effect of the pure Co SACs (98.4 times). The synergetic dual-site atomic catalysts contributed to accelerating the decomposition of H2O2 into singlet oxygen as well as superoxide radical anions to display superb catalytic performances. For a concept employment, Co3N@Co SACs were attempted to utilize as CL probes for establishing a sensitive immunochromatographic assay to quantitate pesticide residues, in which imidacloprid was adopted as the model analyte. The quantitative range of imidacloprid was 0.05–10 ng mL–1 with a detection limit of 1.7 pg mL–1 (3σ). Furthermore, the satisfactory recovery values in mock herbal medicine samples demonstrated the effectiveness of the proposed Co3N@Co SAC-based CL platform. In the proof-of-concept work, synergetic dual-site atomic catalysts show great perspectives on trace analysis and luminescent biosensing.