Nanomaterials with enzyme‐mimicking characteristics have engaged great awareness in various fields owing to their comparative low cost, high stability, and large‐scale preparation. However, the wide application of nanozymes is seriously restricted by the relatively low catalytic activity and poor specificity, primarily because of the inhomogeneous catalytic sites and unclear catalytic mechanisms. Herein, a support‐sacrificed strategy is demonstrated to prepare a single iron site nanozyme (Fe SSN) dispersed on the porous N‐doped carbon. With well‐defined coordination structure and high density of active sites, the Fe SSN performs prominent peroxidase‐like activity by efficiently activating H2O2 into hydroxyl radical (•OH) species. Furthermore, the Fe SSN is applied in colorimetric detection of glucose through a multienzyme biocatalytic cascade platform. Moreover, a low‐cost integrated agarose‐based hydrogel colorimetric biosensor is designed and successfully achieves the visualization evaluation and quantitative detection of glucose. This work expands the application of single‐site catalysts in the fields of nanozyme‐based biosensors and personal biomedical diagnosis.