We developed the functionalized upconversion nanoprobe with ultrasensitive and responsive properties for glucose detection, the responsive emission and satisfactory differentiated performances are observed in situ and in vitro. Display omitted •Ultrasensitive and rapid detection strategy of glucose was constructed based on lanthanide-doped upconversion nanoprobe.•The functionalized nanoprobe can distinguish cancer cells from normal cells by in vitro emission intensity via optical bioimaging.•The designed nanoprobe would facilely achieve glucose detection in situ and in vitro with ultralow detection limit. Various approaches for detecting glucose concentration in real time are emerging at a breakneck pace. Glucose metabolism is closely linked to severe pathological events, which would either cause or predispose many progressive diseases in human. Herein, hydrophilic upconversion nanoprobes NaGdF4: Yb3+, Er3+@Ag anchored with glucose oxidase (GOx) for glucose detection with lower detection limits have been efficaciously constructed. In the upconversion nanoprobes, NaGdF4: Yb3+, Er3+ cores, and Ag layers act as energy donors and effective quenchers, respectively, through energy transfer. Moreover, the layer of Ag may disintegrate by H2O2 in the presence of glucose when glucose oxidase anchoring on the exterior of NaGdF4: Yb3+, Er3+@Ag nanoprobes, which leads to the phenomenon of upconverting emission recovery. Additionally, NaGdF4: Yb3+, Er3+@Ag-GOx has an ultralow detection limit of 1.77 μmol L−1 on glucose detection, and it can achieve optical bioimaging to distinguish cancer cells from normal cells. As a result, the NaGdF4: Yb3+, Er3+@Ag nanoprobes could be expanded to detect diverse H2O2-involved analytes. Overall, this nanoprobe has promising potential to be a compelling tool for the biomedical applications.