In the optimization process of the routes of customized buses, there are numerous uncertainties in the route planning and setting. In this study, the uncertainty theory is introduced into the optimization problem of a customized bus route, and an uncertain customized bus route optimization model is established, which aims at the minimizing the total mileage of vehicle operation. An improved genetic algorithm is used to solve the model, whose feasibility is verified by a case study. The results show that the optimization model based on the uncertainty theory can yield a reasonable customized bus route optimization scheme, and the total mileage reduced from 35.6 kilometers to 32.2 kilometers. This research provides the theoretical support for the optimization of customized bus routes.