Background: Lugol's iodine staining (LIS) solution is used to assist in esophageal chromoendoscopy. Mucosal irritation often results in uncomfortable symptoms and the risk of allergy. In the present study, we developed an extremely low-irritation, easy-to-use mucosal staining solution using polyvinyl pyrrolidone-iodine (PVP-I). This study aimed to evaluate its mucosal staining capacity and biocompatibility. Methods: The mucosal staining capacity was evaluated using postoperative esophageal endoscopic submucosal dissection (ESD) specimens. After elution with vitamin C solution, the specimens were sprayed with LIS as a self-control. The effects on the "lesion area" outlined between the innovative staining and LIS were examined by the intraclass correlation coefficient (ICC) and rank-sum test. To evaluate the biocompatibility, esophageal perfusion was performed with the mice, and the esophagus and stomach were anatomized at different time points for pathological analysis. Results: A total of 53 esophageal ESD specimens were stained at the endoscopy center of West China Hospital. Compared with the same concentration of LIS, the PVP-I solutions showed similar staining effects in terms of the lesion area outlined [ICC = 0.998 (> 0.75), P = 0.733 (> 0.05)]. In live mouse experiments with esophageal perfusion, PVP-I was safer and less irritable to the esophageal gastric mucosa than LIS was, with a lower mucosal damage index (UI) and fewer inflammatory cells in pathological analysis at various time points. Conclusions: The innovative PVP-I staining demonstrated effectiveness, safety, and feasibility as a mucosal staining solution for screening esophageal lesions. Further clinical studies will be carried out in the future.