Purpose To analyse the factors associated with rapid and significant improvement in visual acuity in patients with Leber's hereditary optic neuropathy (LHON) after gene therapy and explain the theory of this improvement. Methods We recruited 149 patients with LHON, who underwent gene therapy, and divided them into two groups according to the absence or presence of rapid and significant visual acuity improvements within 3 days of treatment. A bivariate logistic regression model was used to analyse relevant factors including age, the period between onset and treatment, baseline values of best corrected visual acuity (BCVA), visual field index (VFI) and pretreatment average retinal nerve fibre-layer thickness (GRNFL). The corresponding parameters for the injected and non-injected eyes were analysed separately. Results The period between onset and treatment, and pretreatment baseline BCVA was significantly associated with rapid and significant improvement in visual acuity for both, the injected and non-injected eyes. Moreover, pretreatment baseline VFI and GRNFL were not significantly associated with rapid and significant improvement in visual acuity. Age was significantly associated with rapid and significant improvement in visual acuity of the injected eyes. Conclusion The period between onset and treatment, and pretreatment baseline BCVA may be important predictors of rapid and significant improvement in visual acuity in patients with LHON after gene therapy.