Abstract: Objective To investigate the characteristics and interocular symmetry of retinal nerve fiber layer (RNFL) thickness by spectral-domain optical coherence tomography (SD-OCT) in healthy subjects. Methods The RNFL thickness was measured in 217 subjects (434 eyes) admitted to Yan'an People's Hospital from October to December in 2015 using SD-OCT. Each eye had circular scans performed around the optic nerve, with a circle of 3.4 mm diameter. The average RNFL thickness and mean RNFL thickness in each quadrant were recorded. Pearson correlation analysis was performed to assess the relationships between RNFL thickness and other potential factors. The RNFL thickness was compared between genders. Interocular differences (right eye minus left eye) in the global area and quadrant RNFL thicknesses were measured. Results The average RNFL thickness in 434 eyes was (100.41±9.44) μm. The mean RNFL thickness measurements at superior, nasal, inferior and temporal quadrants in the study population were (120.94±19.81) μm, (65.55±11.55) μm, (126.18±17.32) μm, and (89.10±17.26) μm, respectively. Significant differences were detected among different quadrants (P＜ 0.001). Univariate linear regression analysis showed that RNFL thickness was significantly correlated with age, gender, spherical equivalent, and axial length, while, multivariate regression analysis showed that age and axial length were independently related to RNFL thickness in different quadrants. Mean interocular RNFL thickness differences in the global area and in the superior, nasal, inferior, and temporal quadrants were 0.90 μm (P=0.047), -7.08 μm (P＜ 0.001), 4.47 μm (P＜ 0.001), 2.47 μm (P=0.006), and 3.61 μm (P＜ 0.001), respectively. Conclusion The RNFL thickness is inferior＞ superior＞ temporal＞ nasal in healthy subjects. RNFL thickness decreases with age. Significantly interocular differences can be found in RNFL thickness in healthy eyes.