Objective The purpose was to determine the diagnostic yield of Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in evaluating gastrointestinal lesions with inconclusive endoscopic biopsies. Methods A retrospective analysis was performed in 65 patients who were found to have gastrointestinal lesions with inconclusive endoscopic biopsies and underwent EUS-FNA in our hospital. Compared with surgical histopathology and follow-up status, the utility of EUS-FNA in evaluating such lesions was investigated. Results This study included 41 males (63%) and 24 females (37%) with a median age of 60.0 years. The most common endoscopic appearances were diffuse infiltrative lesions (56.9%), followed by submucosal protrusion types (26.2%). Fifty-four cases (83.1%) were shown to have malignant lesions, and 11 cases (21.7%) were shown to be benign. The overall sensitivity, specificity, and accuracy of EUS-FNA for gastrointestinal lesions with inconclusive biopsies were 76.8% (95%CI: 65.7%-87.8%), 100% (95%CI: 66.4%-100%), and 80.0% (95%CI: 70.3%-89.7%), respectively. For diffuse infiltrative lesions, the sensitivity, specificity, and accuracy of EUS-FNA was 70.6% (95%CI: 55.3%-85.9%, 100% (95%CI: 29.2%-100%), and 73.0% (95%CI: 58.7%-87.3%), respectively. For submucosal protrusions, the sensitivity, specificity, and accuracy of EUS-FNA was 68.8% (95%CI: 46.0%-91.5%), 100% (95%CI: 2.5%-100%), and 73.0% (95%CI: 44.0%-89.7%), respectively. Conclusion EUS-FNA had a moderate diagnostic value in diagnosing endoscopic biopsy-inconclusive gastrointestinal lesions. It can be an alternative option when standard methods, such as endoscopic mucosal forceps biopsy, failed to provide a definitive diagnosis. Complementary techniques and additional refinements in EUS-FNA may enhance its diagnostic efficiency in such lesions.