Recently, our group reported a chemical timer approach to manipulate the onset time of chemiluminescence (CL) emission. However, it is still in the proof-of-concept stage, and its analytical applications have not been explored yet. Nanomaterials have merits of good catalytic effect, large specific surface area, good biocompatibility, and ease of self-assembly, which are ideal for constructing analytical-interfaces for bioassays. Herein, an emission onset time-adjustable chemiluminescent L012-Au/Mn2+ was synthesized for the first time by modifying Mn2+ on the surface of L012-protected gold nanoparticle. By using H2O2 and NaHCO3 as coreactants, L012-Au/Mn2+ could not only generate an ultrastrong and long-time CL emission but also its CL emission onset time could be adjusted by the addition of thiourea, which could effectively eliminate interference from the addition of coreactants, shorten the exposure time, reduce the detection background, and finally achieve high sensitivity CL imaging analysis. On this basis, a label-free CL immunoassay was constructed with a smartphone-based imaging system for high-throughput and sensitive determination of severe acute respiratory syndrome coronavirus 2 nucleocapsid (N) protein. The CL image of the immunoassay with different concentrations of N proteins was captured in one photograph 100 s after the injection of H2O2 with a short exposure time of 0.5 s. The immunoassay showed good linearity over the concentration range of 1 pg/mL to 10 ng/mL with a detection limit of 0.13 pg/mL, which was much lower than the reported CCD imaging detection method. In addition, it showed good selectivity and stability and was successfully applied in serum samples from healthy individuals and COVID-19 rehabilitation patients.