Interfacial hydrogelation of peptides gated on the semiconductor channel has yet to be explored in surface chemistry and field-effect transistor-based biosensors (bio-FETs). This work reports the first example of peptidic hydrogel gated bio-FET by surface assisted self-assembly for label-free and real-time monitoring of the glucose with low limit of detection, excellent sensitivity, and good stability in mouse serum. The boric acid-containing peptide forms a hydrogel layer on the surface of In₂O₃, which interacts with glucose reversibly and generates boronate anions to influence the semiconductor's conductivity. The results also indicate the superiority of hydrogel gated bio-FET for real-time monitoring of glucose in complex environments and serve as a wearable device. This work illustrates a simple and fundamental strategy for integrating peptidic hydrogel with bio-FET for the real-time detection of analytes.