Objective To investigate the value of fluorescin-aided confocal laser endomicroscopy (CLE) for diagnosis of helicobacter pylori (Hp) infection. Methods From June 2009 to November 2009, patients undergone gastric endoscope examination with upper gastrointestinal symptoms (upper abdominal discomfort, abdominal distension, satiation, acid reflux and eructation) or screened for gastric cancer were enrolled. The gastric mucosa CLE image data of twenty diagnosed Hp positive patients and 10 Hp negative patients was analyzed retrospectively. By comparing with histological image of targeted biopsy tissue, the CLE diagnostic criteria for Hp infection were established. In the prospective study, CLE diagnose result was compared with Hp tested result. The consistency of CLE diagnostic criteria in different observers was also analyzed. The CLE image data with histopathology result were compared accordingly. Results Total 72 patients were enrolled in the prospective study,of 34 Hp positive patients, 31 patients were correctly diagnosed by CLE. The accuracy, sensitivity and specificity of CLE diagnosis were 88.9%, 91.2 and 86.8% respectively. CLE image displaying fluorescin leakage and cell shedding was the highest specificity for Hp infection diagnosis, (97.4 %);fluorescin leakage plus gastric pits distortion and cell edema was the highest sensitivity (88. 2%). The consistency of CLE diagnostic criteria in different observers was high (Kappa value 0. 72, 0.87). The CLE image of Hp infection was highly correlated with inflammation activity (P＜0. 001). Conclusion CLE can accurately distinguish normal mucosa from Hp infected mucosa at the cellular level. The diagnostic value for Hp infection was reliable.