ObjectiveTo assess rapid on-line evaluation (ROLE) of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for pancreatic masses. MethodsData of patients with solid pancreatic masses who underwent EUS-FNA in Peking University First Hospital from October 2017 to October 2019 were retrospectively analyzed. Patients were divided into the ROLE group and the control group. The number of FNA passes in ROLE group was decided by the cytopathologist based on the number of pancreatic tissue cells or heteromorphic cells obtained, while that in the control group was decided by the endoscopy physician according to the visual observation of tissue strips. The cytological and histological diagnosis of EUS-FNA specimen were made by another cytopathologist. The gold standard for final diagnosis was based on the histological diagnosis of surgically resected specimens. For patients who did not undergo surgical treatment, the final diagnosis was made based on the clinical course, imaging, tumor markers and feedback on treatment. The diagnostic yield, the accuracy, the sensitivity, the specificity, FNA passes, puncture time and adverse events were compared between the two groups. ResultsA total of 87 patients were enrolled, 51 in the ROLE group and 36 in the control group. The diagnostic yield of the ROLE group was higher than that of the control group without significant difference [98.0% (50/51) VS 86.1%(31/36), P=0.078]. The number of FNA passes in the ROLE group was significantly less than that in the control group (median 2 VS 3， P＜0.001). No significant difference in mean FNA puncture time was found between the ROLE group and the control group (19.4 min VS 18.5 min， P=0.089). There were no significant differences between the two groups with regard to the diagnostic yield, the sensitivity, the specificity, the positive predictive value, the negative predictive value or the incidence of adverse events. ConclusionROLE of EUS-FNA for pancreatic solid tumor may improve tissue acquisition for puncture, reduce the number of FNA passes, and may not significantly increase the procedure time and risk of puncture.