The diagnosis of pancreatic cystic lesions remains challenging. This study aimed to investigate the diagnostic ability of carcinoembryonic antigen (CEA), cytology, and artificial intelligence (AI) by deep learning using cyst fluid in differentiating malignant from benign cystic lesions. We retrospectively reviewed 85 patients who underwent pancreatic cyst fluid analysis of surgical specimens or endoscopic ultrasound-guided fine-needle aspiration specimens. AI using deep learning was used to construct a diagnostic algorithm. CEA, carbohydrate antigen 19-9, carbohydrate antigen 125, amylase in the cyst fluid, sex, cyst location, connection of the pancreatic duct and cyst, type of cyst, and cytology were keyed into the AI algorithm, and the malignant predictive value of the output was calculated. Area under receiver-operating characteristics curves for the diagnostic ability of malignant cystic lesions were 0.719 (CEA), 0.739 (cytology), and 0.966 (AI). In the diagnostic ability of malignant cystic lesions, sensitivity, specificity, and accuracy of AI were 95.7%, 91.9%, and 92.9%, respectively. AI sensitivity was higher than that of CEA (60.9%, p = 0.021) and cytology (47.8%, p = 0.001). AI accuracy was also higher than CEA (71.8%, p < 0.001) and cytology (85.9%, p = 0.210). AI may improve the diagnostic ability in differentiating malignant from benign pancreatic cystic lesions.