The rational construction of nanomaterials with boosted peroxidase (POD)-like activity is momentous in artificial enzyme design and biological catalytic fields. Herein, a hybrid nanozyme, gold nanoparticles/N-doped porous carbon (AuNPs/ NPC), is fabricated via a supramolecular assembly-assisted pyrolysis strategy and engineered as a peroxidase mimic. In this strategy, a melamine-cyanurate supramolecular aggregate can be employed not only as a self-vanishing template to gain porous morphology but also as a nitrogen source to achieve an exceptional high N doping. The obtained NPC is then subsequently used to immobilize AuNPs via an in situ reduction approach. Benefiting from well-dispersed ultrafine AuNPs, high N content, hierarchical porous architecture, and the synergistic effect of AuNPs and NPC, the fabricated nanozyme exhibits enhanced POD-like activity, making it a potential alternative to peroxidase mimics. Besides, the AuNPs/NPC shows highly electrocatalytic properties, which could serve as a signal amplification platform for ultrasensitively detecting hydrogen peroxide (H 2 O 2 ). The hybrid nanozyme-based electrochemical sensor shows a linear relationship within 0.2− 7000 μM. Significantly, the sensitivity and limit of detection of the fabricated sensor are 285.9 μA mM −1 cm −2 and 67 nM, respectively. Also, this biosensor is applied to detect H 2 O 2 in human serum samples and A549 cells with desirable results. Therefore, the present work offers a facile strategy for the fabrication of a high N-contained hybrid nanozyme to simulate the catalytic activity of natural enzymes and exhibits broad prospects in biosensing, mimicking-enzyme catalytic fields, and clinical diagnosis.