Background: In cases of Hirschsprung disease, complete and reproducible resection of the aganglionic bowel is ideal to achieve good postoperative bowel function. Reliable identification of the upper margin of the surgical anal canal, which is the squamous-columnar junction, is necessary during transanal pull-through. Here, we describe a novel staining technique using Lugol’s iodine stain to visualize the upper margin of the surgical anal canal. Methods: Lugol’s iodine staining was performed in five patients with Hirschsprung disease treated using a single-stage laparoscopic transanal pull-through modified Swenson procedure. In two of these patients, endocytoscopic observation with ultra-high magnification was performed using methylene blue and crystal violet to mark the border of the squamous epithelium at 1 week before surgery. The alignment between the incisional line, which was revealed using Lugol’s iodine staining and endocytoscopic marking, was evaluated. Complications, including postoperative bowel dysfunction, were evaluated. Results: In all cases, Lugol’s iodine staining produced a well-demarcated line. The endocytoscopic marking of the upper margin of the surgical anal canal was aligned with the line revealed by Lugol’s iodine staining. There were no complications associated with the transanal pull-through procedure, including postoperative bowel dysfunction. Conclusions: Lugol’s iodine staining could be a safe and practical method to visualize the upper margin of the surgical anal canal intraoperatively. This finding may be useful for surgeons to make a consistent removal of the aganglionic bowel during surgery for Hirschsprung disease.