As a H2O2‐dependent bioluminescent substrate, tetrabromo‐1,4‐benzoquinone (TBBQ) was first isolated from acorn worm. The mechanism of chemiluminescence (CL) corresponding to the bioluminescence (BL) of acorn worm is largely unknown, let alone the mechanism of BL. In this article, we firstly studied the chemical and physical processes, and mechanism of H2O2‐dependent CL from TBBQ by theoretical and experimental methods. The research results indicate: the CL process is initiated by a nucleophilic substitution reaction, which leads to the formation of an anionic dioxetane through five consecutive reactions; the anionic dioxetane decomposes to the first singlet excited state (S1) via a conical interaction of the potential energy surfaces (PESs) between the ground (S0) and S1 state; the anionic S1‐state changes to its neutral form by a proton transfer from the solvent and this neutral product is assigned as the actual luminophore. Moreover, the experimental detection of CL, .OH and the identifications of 2,3‐dibromo maleic acid and 2‐bromo malonic acid as the major final products provide direct evidence of the theoretically suggested mechanism. Finally, this study proves that the activity of the H2O2‐dependent CL from TBBQ is significantly lower than the one from tetrachloro‐1,4‐benzoquinone (TCBQ), which is caused by the weaker electron withdrawing effect and the stronger heavy atomic effect of bromine.