Vanin-1 is a pantetheine hydrolase that plays a key role in inflammatory diseases. Effective tools for noninvasive, real-time monitoring of Vanin-1 are lacking, largely due to background fluorescence interference in existing probes. To address this issue, we developed a dual-modal fluorescent and colorimetric probe, MB-Van1, to detect Vanin-1 with high sensitivity and selectivity. MB-Van1 has a structure optimized to exhibit nearly zero background fluorescence, resulting in a high signal-to-noise ratio that enables the accurate detection of Vanin-1 activity in various biological tissues. In vitro experiments demonstrated that MB-Van1 had a detection limit as low as 0.031 ng/mL in the fluorescence mode. We successfully employ MB-Van1 to observe elevated Vanin-1 levels in inflammatory tissues of various mouse models of rheumatoid arthritis (RA), drug-induced liver injury (DILI), and nonsteroidal anti-inflammatory drug (NSAID) enteropathy models, within only 5 min. This advancement provides a novel approach for monitoring the dynamic changes of Vanin-1 during inflammation, offering new strategies for the early diagnosis and therapeutic assessment of other related diseases.