Objective To study the relationship between retinal nerve fiber layer (RNFL) thickness measured by optical coherence tomography (OCT) and visual function, disease course and system dysfunction in patients with multiple sclerosis (MS); to evaluate the significance of RNFL thickness as a biological marker for axonal loss in the course of MS disease. Methods This was a cross-sectional study. Seventeen MS patients (32 eyes) and 17 healthy people (17 eyes) were matched by age and sex. All participants received a neurological evaluation and a complete ophthalmological examination, including visual acuity, OCT measurement of RNFL thickness, pattern visual evoked potentials (PVEP) and electroretinography (ERG) examinations. Patients were divided into three groups: MS patients with a history of optic neuritis (MS-ON group), MS patients without a history of optic neuritis (MS-NON group) and the control group. The results of OCT measurement of RNFL thickness, and PVEP and ERG examinations of the three groups were compared by ANOVA. The relationship between RNFL thickness and the score on the expanded disability status scale (EDSS), and disease course, and electrophysiology results were analyzed by Spearman correlation analysis. Results The MS-ON eyes showed RNFL thinning in the average, superior, inferior, nasal, and temporal quadrants [(72.4±16.6), (80.5±26.3), (84.2±29.5), (65.8±14.0), (54.2±16.4)μm] compared to the control eyes [(105.6±10.8), (119.7±18.2), (123.5±17.9), (91.1 ±21.2), (88.2±13.0)μm] (P=0.000 each). The differences in RNFL thickness in the average, superior, inferior, and temporal quadrants between MS-ON eyes and MS-NON eyes [(98.3±12.0), (115.5±18.2), (117.7±21.5), (72.0±15.0), (68.1 ±16.1 )μm] were significant (P＜0.05 each), while the nasal quadrant did not show significant differences. Compared to control eyes, RNFL in the nasal and temporal quadrants in MS-NON eyes showed a significant reduction (P＜0.05 each), while the average, superior and inferior quadrants did not show significant thinning. RNFL thickness in MS-ON eyes was significantly correlated with the disease course of MS [(8.8±9.8)year] (r=-0.659, P=0.028) and EDSS scores (2.9±2.2) (r=-0.741, P=0.046). In this research, no significant relationships were found between RNFL thickness and disease course or EDSS scores in the MS-NON group. RNFL thickness in MS eyes showed a significant relationship to the latency and amplitude of P100 wave of both the 60' and 15' angles (r=-0.416, 0.332,-0.317, 0.265, P＜0.05 each), and the latency and amplitude of the a wave and latency of the b wave in the ERG maximum response (r=-0.471, 0.415,-0.360, P＜0.05 each), while no relationship was found between RNFL thickness and the amplitude of the b wave. Conclusion RNFL thickness measured by OCT can be used for clinical application as a structural biomarker of axonal loss in MS patients.