Designing unique nanomaterials for the selective sensing of biomolecules is of significant interest in the field of nanobiotechnology. In this work, we demonstrated the synthesis of ordered and Cu nanoparticles functionalized mesoporous C3N5 with unique peroxidase like nanozymatic activity for the ultrasensitive and selective detection of glucose and glutathione. A nano hard‐templating technique together with the in‐situ polymerization and self‐assembly of Cu and high N‐containing CN precursor was adopted to introduce mesoporosity as well as high N and Cu content in mesoporous C3N5. Due to the ordered structure and highly dispersed Cu in the mesoporous C3N5, a large enhancement of the peroxidase mimetic activity in the oxidation of a redox dye in the presence of hydrogen peroxide could be obtained. Additionally, the optimized Cu functionalized mesoporous C3N5 exhibited excellent sensitivity to glutathione with a low detection limit of 2.0 ppm. The strong peroxidase activity of the Cu functionalized mesoporous C3N5 was also effectively used for the sensing of glucose with the detection limit of 0.4 mM through glucose oxidation with glucose oxidase. This unique Cu functionalized mesoporous C3N5 has the potential for detecting various molecules in the environment as well as for next‐generation glucose and glutathione diagnostic devices.