Highly sensitive light-up near-infrared fluorescent probe for detection and imaging of β-glucuronidase in human serum, living cells and tumor-bearing mice

β-Glucuronidase (GUS) plays a key role in tumor initiation, metastasis, and progression, thus, has been proposed as promising cancer biomarker. In this study, we designed an enzyme-activatable near-infrared (NIR) fluorescent probe (DCM-βGlcA) for the rapid accurate detection of GUS activity vitro, vivo ex vivo. The DCM-βGlcA was prepared by linking glucuronic acid residue to dicyanomethylene-4H-pyran (DCM). This exhibited significant light-up NIR signals at 680 nm after reacting with Stokes shift could reach 150 nm. showed high sensitivity toward excellent linear relationship concentrations ranging between 0 4 U L−1 (R2 = 0.9974) limit low 0.19 L−1. We used identify serum levels both patients healthy individuals similar accuracy that enzyme-linked immunosorbent assay (ELISA) while being easier faster perform. Moreover, tracking endogenous living cells, thereby discriminating GUS-overexpressed liver from normal cells. Additionally, able detect image tissue tumor-bearing mouse models. These findings demonstrate potential tool detecting monitoring preclinical applications.