Herein, we have developed a simple and facile method to synthesize yolk–shell nanostructured Fe₃O₄@C nanoparticles (NPs) as a multifunctional biosensing platform for the label-free colorimetric detection of H₂O₂ and glucose. It was demonstrated that Fe₃O₄@C yolk–shell nanostructures (YSNs) retained the magnetic properties that can be used for separation and concentration. Also importantly, the Fe₃O₄@C YSNs exhibited an intrinsic peroxidase-like activity that could quickly catalyze the enzyme substrate in the presence of H₂O₂ and produce a blue color. Compared to other similar ferric oxide-based NPs with different structures, Fe₃O₄@C YSNs exhibited greatly enhanced catalytic activities due to their unique structural features. Moreover, steady-state kinetics indicated the catalytic behaviors in agreement with the classic Michaelis–Menten models. Taking advantage of the high catalytic activity, Fe₃O₄@C YSNs were employed as novel peroxidase mimetics for label-free, rapid, sensitive, and specific colorimetric sensing of H₂O₂ and glucose, suggesting that Fe₃O₄@C YSNs have the potential for construction of portable sensors in the application of point-of-care (POC) diagnosis and on-site tests.