beta-Glucuronidase (GUS) is an acidic hydrolase enzyme overexpressed in various inflammatory diseases, making it a promising biomarker for inflammation. However, current tools for real-time, in situ imaging of GUS activity are hindered by background interference, which reduces their effectiveness in dynamic biological environments. To address this challenge, we developed Ox-GUS, a GUS-specific fluorescent probe with a unique molecular design featuring a disrupted conjugated structure. This design provided Ox-GUS with near-zero background optical properties, a significantly enhanced signal-to-noise ratio, and a highly sensitive detection ability. The probe demonstrated a fluorescence enhancement of up to 400 folds in response to GUS activity, with a detection limit as low as 0.0035 U/mL. We successfully employed Ox-GUS to visualize GUS activity in real-time in mouse models of rheumatoid arthritis, autoimmune hepatitis, and inflammatory bowel disease, and effectively monitored therapeutic responses. This study highlights the potential of Ox-GUS as a robust tool for advancing research on GUS-related inflammatory mechanisms and for early diagnosis and treatment monitoring of inflammatory diseases.