To alleviate the problems of traffic congestion, excessive energy consumption, and the environmental pollution caused by private cars, it is essential to use public transportation (PT). However, passengers making multiple trips in a short time period must repeatedly make travel mode choices, purchase tickets, and wait for buses for each trip, which may negatively affect their preference for PT. In order to improve the attractiveness of PT, especially for passengers requiring multiple trips in a short time period, this paper proposes the one e-ticket customized bus service mode for passengers with multiple trips (OECBSM-PMT) by customized buses (CBs). Besides, a CB-routing optimization model for the OECBSM-PMT is also developed in this paper, formulated as a mixed-integer linear programming based on a vehicle routing problem with pickup and delivery and time windows (VRPPDTW). The model aims to maximize the profit and minimize the costs of operation with considering passengers with multi-trip requests, homogeneous CB fleets with pickup/delivery-time-window constraints, and mixed loads. A service effectiveness identification procedure based on genetic algorithm (GA) is proposed to cope with the calculation considering the characteristics of passengers with multiple trips. Finally, the proposed model and algorithm are verified and analyzed using the case of the 2022 Beijing Winter Olympic Games. It can be found from the results that the method can provide an optimized CB route plan and timetable, and the algorithm GA-I obtains better solutions than other solving strategies in most instances. The proposed OECBSM-PMT and corresponding optimized method can better adapt to diverse travel demands, significantly improve the convenience for passengers, especially those making multiple trips in a short time period and will eventually promote a higher level of public transport service.