Pantetheinase, a glycosylphosphatidylinositol (GPI) anchored enzyme, overexpresses in intestine, liver, and kidney with various biological functions such as its linkage to the inflammation and some metabolic diseases. It can hydrolyze pantetheine to cysteamine, an antioxidant, and pantothenic acid (Vitamin B5) that is an essential component of coenzyme A (CoA). Until now, very few analytic methods were developed for this enzyme, hampering the further investigation of its biological functions. In this work, we report the design, synthesis, and biological examination of a highly sensitive bioluminogenic probe for pantetheinase with a limit of detection of 1.14 ng/mL. Furthermore, animal experiments validated that our probe can be applied to detect the endogenous pantetheinase activity. To the best of our knowledge, this is the first bioluminogenic probe achieving the detection of pantetheinase level in vivo.