OBJECTIVE: To apply the measurement of changes in the degree of eyelid ptosis in myasthenia gravis (MG) after neostigmine injection to provide an objective, precise and standardized measure for clinical diagnosis. METHODS: This study included 34 MG cases, 30 non-MG patients, and 30 normal healthy individuals. Before and after the neostigmine injection test, the lid size of patients in the MG group, non-MG control group and normal control group was measured, and the relative values at multiple measurements and manifestation were analyzed in the MG and control groups, and the sensitivity, specificity and Jorden index of the multiple measurements were calculated. RESULTS: The mean ± standard deviation of the multiple measurements of the flat lid fissure size in patients in the MG group, the non-MG control group and the normal control group were 2.621 ± 0.376 mm, 0.823 ± 0.266 mm and 0.853 ± 0.254 mm, respectively. the differences between the MG group and the non-MG and normal groups were statistically significant (P < 0.05). The sensitivity, specificity, and Jorden index of the multiple measurement values were 96.7%, 100%, and 0.967, respectively. CONCLUSION: The application of the video ophthalmometry system for the neostigmine test to assist in the diagnosis of MG is an objective, accurate, and standardized measurement method.