Interface stiffness is an important factor influencing the performance of mechanical products. Uncertain factors affect the interface stiffness and stability in the process of product design, manufacture, and operation. How to reduce the impact of uncertain factors on the interface stiffness is a vital problem in interface design. In this paper, a robust optimal design method is proposed for mechanical interfaces considering uncertain factors, which combines the finite element simulation, experiment, and optimization to reduce the sensitivity of interface stiffness to uncertain factors. The proposed interface design method provides an effective way to improve the interface stiffness under uncertain conditions. In order to validate the proposed method, the bolted connection structure of a flange is applied as an example. The interface stiffness of the flange is selected as an optimization target, and the Gaussian process regression is used to construct a two-layer optimal model of the objective function for the design and uncertain parameters. When experimental and optimization results differ significantly, the Kalman filter is used to provide the feedback for the optimization results until the results meet requirements. The final results show that the optimized mechanical interface stiffness is increased by 15.5%, and the error between the optimized prediction and experimental results is within 1% after three times experimental validation and feedback adjustment.