Herein, we report the soft-templated preparation of mesoporous iron oxide using an asymmetric poly(styrene-b-acrylic acid-b-ethylene glycol) (PS-b-PAA-b-PEG) triblock copolymer. This polymer forms a micelle consisting of a PS core, a PAA shell, and a PEG corona in aqueous solutions, which can serve as a soft template. The mesoporous iron oxide obtained at an optimized calcination temperature of 400 °C exhibited an average pore diameter of 39 nm, with large specific surface area and pore volume of 86.9 m2 g–1 and 0.218 cm3 g–1, respectively. The as-prepared mesoporous iron oxide materials showed intrinsic peroxidase-like activities toward the catalytic oxidation of 3,3′,5,5′-tertamethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). This mimetic feature was further exploited to develop a simple colorimetric (naked-eye) and electrochemical assay for the detection of glucose. Both our colorimetric (naked-eye and UV–vis) and electrochemical assays estimated the glucose concentration to be in the linear range from 1.0 μM to 100 μM with a detection limit of 1.0 μM. We envisage that our integrated detection platform for H2O2 and glucose will find a wide range of applications in developing various biosensors in the field of personalized medicine, food-safety detection, environmental-pollution control, and agro-biotechnology.