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Objective To evaluate the sensitivity and specificity of Rome Ⅲ criteria in diagnosis of functional dyspepsia (FD) and assess its value in differentiate FD from other organic diseases in upper gastrointestinal tract. Methods Four thousand nine hundred and sixty-two patients, who underwent gastroscopy from July to August 2006 and March to April 2007, were consecutively enrolled and interviewed face to face with a standard questionnaire. The patients who were diagnosed as FD were according to Rome Ⅲ criteria, and those who were diagnosed as upper gastrointestinal diseases, such as chronic erosive gastritis (CEG), gastric ulcer (GU), duodenal ulcer (DU) and gastric carcinoma (GC), were done by gastroscopy and pathology. The differences of clinical characteristics among these diseases were analyzed. Results The FD patients accounted for 7.58% (376/4962) with female in predominant (P = 0.000). The patient with CEG, GU, DU or GC accounted for 29.99% (1488/4962), 1.89% (94/4962),4.25% (211/4962) or 4.57% (227/4962), respectively, all with male in predominant (P<0.05). Further analysis indicated that the age of onset between patients with FD and DU was no difference, but was younger than those with other three diseases (P<0.05). The incidence of early satiation and postprandial in FD patients were higher than those in other patients (P<0.05). The incidence of belching, nausea and vomiting showed no difference among these patients(P>0.05). According to Rome Ⅲ criteria, the symptoms of epigastric pain, early satiation, postprandial fullness and epigastric burning had higher sensitivity and specificity (except epigastric burning)in diagnosing FD (P<0.05), with highest Youden index in epigastric pain (0.42) and postprandial fullness (0.46). Conclusion Rome Ⅲ criteria has high specificity and sensitivity in diagnosing FD, and also has an important value in differentiate FD from other organic diseases.