Abstract-Objective: aiming to establish a new strategy based on uniform design and genetic algorithm and applying in the optimization of the separation condition of Ginkgo biloba extract using reversed-phase liquid chromatography according to traditional optimization methods. Methods: based on uniform design and genetic algorithm, a new strategy was developed and a software package was developed. The optimization strategy was applied in the optimization of separation condition of Ginkgo biloba extract in reversed-phase chromatography, the volume fraction of acetonitrile and rate of linear gradient-elution were selected as the optimization variables. Results: The optimization of separation condition of Ginkgo biloba extract in reversed-phase chromatography was performed and satisfactory optimal result was obtained as the initial concentration of acetonitrile was 14.4% and the rate of linear gradient-elution was 0.22%, respectively. Conclusion: The results shown that the method using uniform design and genetic algorithm is efficient and it has potential application not only in the optimization of two parameters, but also of much more than two parameters.