Increasing roof albedo (using a “cool” roof) and night ventilation are passive cooling technologies that can reduce the cooling loads in buildings, but existing studies have not comprehensively explored the potential benefits of integrating these two technologies. This study combines an experiment in the summer and transition seasons with an annual simulation so as to evaluate the thermal performance, energy savings and thermal comfort improvement that could be obtained by coupling a cool roof with night ventilation. A holistic approach integrating sensitivity analysis and multi-objective optimization is developed to explore key design parameters (roof albedo, night ventilation air change rate, roof insulation level and internal thermal mass level) and optimal design options for the combined application of the cool roof and night ventilation. The proposed approach is validated and demonstrated through studies on a six-storey office building in Xiamen, a cooling-dominated city in southeast China. Simulations show that combining a cool roof with night ventilation can significantly decrease the annual cooling energy consumption by 27% compared to using a black roof without night ventilation and by 13% compared to using a cool roof without night ventilation. Roof albedo is the most influential parameter for both building energy performance and indoor thermal comfort. Optimal use of the cool roof and night ventilation can reduce the annual cooling energy use by 28% during occupied hours when air-conditioners are on and reduce the uncomfortable time slightly during occupied hours when air-conditioners are off.