The classic luminol-H 2 O 2 chemiluminescence (CL) systems suffer from easy self-decomposition of H 2 O 2 at room temperature, hindering the practical applications of the luminol-H 2 O 2 CL system. In this work, unexpectedly, we found that the carbon vacancy-modified Fe-N-C single atom catalysts (V C -Fe-N-C SACs) can directly trigger a luminol solution to generate strong CL emission in the absence of H 2 O 2 . The Fe-based SACs were prepared through the conventional pyrolysis of zeolitic imidazolate frameworks. The massive carbon vacancies were readily introduced into Fe-N-C SACs through a tannic acid-etching process. Carbon vacancy significantly enhanced the catalytic activity of Fe-N-C SACs on the CL reaction of luminol-dissolved oxygen. The V C -Fe-N-C SACs performed a 13.4-fold CL enhancement compared with the classic luminol-Fe 2+ system. It was found that the introduction of a carbon vacancy could efficiently promote dissolved oxygen to convert to reactive oxygen species. As a proof of concept, the developed CL system was applied to detect alkaline phosphatase with a linear range of 0.005-1 U/L as well as a detection limit of 0.003 U/L. This work demonstrated that V C -Fe-N-C SAC is a highly efficient CL catalyst that can promote the analytic application of the luminol CL system.