In the present study, for the first time, N,N′-di-carboxymethyl perylene diimides (PDI) have been employed to functionalize Fe₃O₄ nanoparticles via a facile route. The as-prepared PDI–Fe₃O₄ nanocomposites have been developed as a novel enzyme mimetic and have demonstrated superior peroxidase-like activity to catalyze the oxidation of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ than that of pure Fe₃O₄ nanoparticles as well as other reported Fe-based peroxidase mimics. In addition, kinetic analysis has revealed that the catalytic reaction was in agreement with the typical Michaelis–Menten kinetics, and the PDI–Fe₃O₄ nanocomposites displayed a higher affinity towards substrates such as H₂O₂ and TMB. Moreover, fluorescence data have indicated that the catalytic behavior can be assessed by the electron transfer ability between H₂O₂ and TMB. Based on the advantages of PDI–Fe₃O₄ nanocomposites, such as better dispersity, easy separation, enhanced catalytic activity, sensitivity and selectivity for colorimetric detection of glucose, a colorimetric sensor was designed and used for the rapid detection of H₂O₂ and glucose in a short time.