A sensitive and rapid method was introduced in this research for the detection of H2O2 based on luciferase mimetic. Luciferase mimetic of bioluminescent earthworm was synthesized using a metal–organic framework (MOF). The luminol was loaded into the chromium terephthalate MOF MIL-101(Cr) pores and applied in the luciferin chemiluminescence (CL) system with an outstanding performance. The samples exhibited prominent catalytic performance of luciferase mimetic in the luciferin chemiluminescence system for H2O2 sensing with high sensitivity in the linear range of 1 × 10− 2– × 10− 10 mol/L and detection limit of 1.251 × 10− 11 mol/L (R2 = 0.9944). The effects of the key factors were explored by response surface methodology (RSM) based on a Box–Behnken design (BBD). Thanks to its features such as low cost, rapid response, and portability, this enzyme mimetic could be a promising platform for CL sensing, offering great potential for the construction of a CL H2O2 sensor for biotechnology and clinical diagnostic purposes