Objective To evaluate the efficacy and safety of SPOT (GI Supply, USA), a new carbon-based permanent marker approved by the Food and Drug Administration (FDA), in the endoscopic marking for gastrointestinal lesions. Methods A total of 115 patients with gastrointestinal lesions who underwent endoscopic treatment or surgery in Beijing Friendship Hospital or Beijing Chao-Yang Hospital from April 2019 to November 2019 were enrolled in the study. SPOT was used to mark the lesions, and marking points were found during endoscopic treatment or surgery to calculate the effective marking rate by single-group target value method. Adverse events after marking were recorded, and the changes of blood routine test, liver and kidney functions before and after marking were compared. Results The effective rate of endoscopic marking with SPOT was 99.13% (114/115). The longest marking time was 57 days. There was no puncture of intestinal wall or injection into abdominal cavity during the marking process. One patient developed mild fever after marking. The incidence of adverse events was 23.48% (27/115), which were all unrelated to the test equipment. There was no significant difference in blood routine tests or liver and kidney functions before and after marking (P>0.05). Conclusion SPOT produced by GI Supply can effectively mark gastrointestinal lesions without serious adverse events, which meets the requirements of clinical use.