Pantetheinase (also known as Vanin-1) is highly expressed in the liver, kidneys, and intestine and is closely associated with a number of diseases. Vanin-1 can hydrolyze pantetheine to pantothenic acid (vitamin B5) and cysteamine and participate in the synthesis of glutathione (GSH). GSH is highly expressed in tumor cells and plays a major role in the resistance of tumor cells to cisplatin. Therefore, we urgently need a method to monitor the activity level of Vanin-1 in tumor cells and tissues and elucidate the relationship between the role of Vanin-1 in GSH synthesis and tumor resistance. Herein, we report a Cy-Pa fluorescent probe for imaging Vanin-1 in cells and in vivo that can qualitatively and quantitatively detect the fluctuation of Vanin-1 concentrations in HepG2 and HepG2/DDP cells or tumor tissues of tumor-bearing mice. This probe shows excellent potential in in situ real-time monitoring of endogenous Vanin-1. Moreover, we proved that Vanin-1 can inhibit GSH synthesis using the probe. When the Vanin-1 inhibitor RR6 was used in combination with cisplatin, HepG2 and HepG2/DDP cells showed increased resistance to cisplatin, while the therapeutic efficiency of cisplatin was reduced in HepG2 and HepG2/DDP xenografts. In this study, Vanin-1 was shown to play an important role in the treatment of cancer, and the study of Vanin-1 may provide an idea for the treatment of cancer in the future.