Vanin-1, a GPI-anchored pantetheinase, has been implicated in various physiological and pathological processes and is a drug target for inflammatory bowel disease (IBD). We developed PHPO, a novel, specific and sensitive fluorogenic biosensor for Vanin-1 and validated its selectivity in VNN1(-/-) mice. Using PHPO, we imaged the endogenous distribution of Vanin-1 in intestinal regions, detected expression changes during organoid differentiation, and visually revealed the activation process of Vanin-1 in the colon under the IBD model, further highlighting its pathological role. Finally, a high-throughput screening method for Vanin-1 regulator was established using PHPO, and identifying OGA (octyl gallate) as a novel inhibitor (IC(50) = 29.9 nM) and elucidating its mechanism via molecular docking. PHPO can thus serve as an innovative tool for Vanin-1 detection, with applications in diagnosis of Vanin-1-related diseases and the potential to guide future therapeutic strategies.