Porous nanomaterials with superior peroxidase mimetic activity (nanozyme) at room temperature have gained increasing attention as potential alternatives to natural peroxidase enzymes. Herein, we report the application of porous iron oxide nanoflakes (IONFs), synthesized using the combination of solvothermal method and high-temperature calcination as peroxidase nanozyme for the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The four IONF catalysts possess porous structures with a wide pore size distribution between 2–30 nm and high specific surface areas around to 200 m2 g−1. The increase of calcination temperature of the IONFs from 250 °C to 400 °C resulted in a gradual decrease in their specific surface area and Michaelis-Menten constant (Km) for TMB oxidation. The optimum IONF sample showed a much lower Km at 0.24 mM (towards TMB) compared to natural enzyme horseradish peroxidase (HRP) at 0.434 mM, revealing the promising potential of the as-prepared IONFs as alternatives to HRP for biosensing applications.