Single atom-dispersed catalysts (SADCs) with highly exposed active sites can be used as sensitive signal probes because of their superior catalytic efficiency. However, the dispersed atoms tend to aggregate, restricting the loading capacity of metal atoms. Herein, the defective sites on Zr-oxo clusters of metal-organic frameworks (MOFs) UiO-66-NH2 were modulated by excessive acetic acid and utilized for confining metal atoms with high loading capacity. To verify the feasibility of the designed strategy, the Co element was loaded onto MOFs UiO-66-NH2 to prepare SADCs with desirable Fenton-like activity. The prepared Co SADCs at a low concentration of 1.0 μg mL-1 are found to boost chemiluminescent (CL) emission for 3700 times due to the significantly improved Co content of 5.55 wt %. The superior CL enhancement efficiency is ascribed to reactive oxygen species generated by the accelerated decay of H2O2. To verify the application potential in CL assay, they were used as signal probes to establish an immunoassay method for carbendazim with a dynamic range of 1.0 pg mL-1 to 25 ng mL-1 and a limit of detection of 0.33 pg mL-1. This defective site modulation strategy paves an avenue for preparing SADCs with a high CL response by improving the loading capacity of metal atoms.