The study of the mechanism of peroxidase-like activity of nanozymes, to obtain high-performance, is of great significance in biosensing and biocatalysis. Herein, the developed bimetal–organic frameworks (FexNiy-MOF) all showed enhanced peroxidase-like activity, and the research was carried out on behalf of Fe3Ni-MOF with the best catalytic activity. The dual mechanism (electron transfer mechanism and generation of hydroxyl radicals (•OH) mechanism) of the enhanced peroxidase-like activity of Fe3Ni-MOF was deduced through the cyclic voltammetry and electron spin resonance. The enhanced peroxidase-like activity mainly depends on the introduction of Ni, which improved the redox capacity of Fe3Ni-MOF and accelerated the electron transfer between TMB and H2O2. In addition, the introduction of Ni also improved the conversion efficiency between Fe3+ and Fe2+, to promote the generation of •OH, and then enhanced the peroxidase-like activity. The Fe3Ni-MOF we obtained can achieve ultrasensitive detection of H2O2 with a linear range of 0.02–15 μM and a detection limit as low as 11 nM. This makes the enhancement of peroxidase-like activity of practical significance. Then, a glucose detection system was successfully established with using H2O2 as an intermediate product, which has great potential in the development of biosensor applications.