Objective To evaluate the accuracy of ^99mTcO4- gastrointestinal imaging in the diagnosis of Meckel’s diverticulum in children. Methods The clinical data of ^99mTcO4- imaging, surgery and pathological results of 95 children with gastrointestinal bleeding were retrospectively analyzed. Results Forty-four cases of 95 patients had positive finding of ^99mTcO4- ectopic gastric mucous membrane imaging, and positive rate was 46.3%. In the positve cases 52.3% cases (23/44) were diagnosed of intestinal heterotopic gastric mucosa, 47.7% cases (21/44) were suspected of intestinal heterotopic gastric mucosa. The sex ratio (males to females) was 7.8:1. Thirty-seven positive cases underwent operation; among them, 35 cases were found to have Meckel’s diverticulum and 1 case had duplication of the digestive tract; Diverticulum lesion was not found in 1 case. The samples of 35 cases diagnosed surgically of Meckel’s diverticulum were examined by pathologic histology. All 35 cases were confirmed as Meckel’s diverticulum and ectopic gastric mucosa epithelium, including 2 cases with ulcer formation, 1 case with small patches of pancreatic tissue, 1 case with ileocecal junction of suppurative enteritis and 1 case with chronic appendicitis. Conclusions 99mTcO4- radionuclide imaging technique is of high value for the non-invasive diagnosis of children’s Meckel’s diverticulum.