Objective To investigate the efficacy of centrally fixed eyeball for assessment of the depth of anesthesia in premature infants undergoing outpatient fundus examination. Methods Fifty eight premature infants undergoing examination of fundus of eyes were enrolled in this study. Their gestational age (from the first day of last menstruation period to birth) + after birth age (from birth to the day when examination of fundus of eyes was performed) = 44-64 weeks. The patients were randomly divided into 2 groups: Ⅰ group body movement (group M, n = 27) and Ⅱ group centrally fixed eyeball (group E, n = 31). Anesthesia was induced and maintained with isoflurane inhalation. The patients were breathing spontaneously. The eyelids were kept open with speculum after induction of anesthesia. The EC50 of sevoflurane concentration which could inhibit body movement or make eyeballs centrally fixed was determined by up-and-down sequential experiment. The initial isoflurane concentration was 3% in both groups. Each time the isoflurane concentration was increased/decreased by 0.5 %. 95 % confidence interval (CI) was calculated. The lowest SpO2, respiratory rate and coughing during maintenance of anesthesia were recorded. Results The EC50 of sevoflurane (95% CI) was 2.9% (2.2%-3.6%) in group M and 3.4%(2.6%-4.6%) in group E. Examination was successfully completed in all patients. No respiratory depression and coughing occurred during examination and no vomiting and coughing were observed during feeding at 1 h after recovery from anesthesia. No body movement occurred in 15 patients whose eyeballs were centrally fixed in group E. Conclusion Centrally fixed eyeball can be used as sign of appropriate depth of anesthesia for fundus examination in premature infants.