An overall framework of the virtual testing system has been established based on the analysis of the virtual testing requirements for autonomous navigation performance of unmanned surface vehicles (USVs). This system consists of several modules, including the environment module, motion module, sensor module, and 3D visualization module. Firstly, within the robot operating system (ROS) environment, a three-dimensional navigation environment was generated by combining actual wave spectra with Gerstner waves. By designing a power plugin for USV navigation, the system was made to reflects the coupled motion model of USVs in wind, waves and currents, along with predictive results. Regarding the four typical sensor information on USVs, the actual sensors were virtualized, and a simulation approach for virtual sensor information is provided. The three-dimensional visualization of USV’s motion enables the intuitive display and analysis of the virtual testing process. Based on the prediction of coupled motion characteristics in wind, waves and currents, the interaction between USVs and the virtual testing system has been realized. A platform for virtual testing experiments to determine the autonomous navigation performance of USVs was established, and the effectiveness of the platform was verified in terms of perception and environmental interference. In virtual environmental interference validation, the average amplitude deviation of the heave motion of USVs under sea state 3 reaches 0.74 m, and the average amplitude deviation of the pitch motion reaches 0.25 rad, showing the gradually increasing disturbance of the sea state. Finally, virtual testing experiments were conducted on a specific USV to evaluate its autonomous navigation perception performance, trajectory tracking performance, and autonomous obstacle avoidance. The evaluation results indicate that the platform can achieve the functionality of virtual testing for the autonomous navigation performance of USVs from the perspective of cost function, taking the reaction distance, regression distance, and obstacle avoidance time into consideration. A representative example is that the cost function deviation rates of overtaking obstacle avoidance between static and dynamic seas reach 5.11%, 8.98% and 18.43%, respectively. The gradually increasing data shows that the virtual simulating method matches the drifting-off-course tendency of boats in rough seas. This includes acquiring perception information of navigation and simulating the motion and navigation processes for visualization. The platform provides new means for testing and evaluating the autonomous navigation performance of USVs.