The universal hinge support (UHS) is a new connection system for the tower and pier of a single-tower cable-stayed bridge (STCSB), which could conform the multi-direction rotation of the tower and release the bending moment at the bottom of the tower in all directions. However, UHS is not an ideal hinge in practical projects, and the rotational constraint stiffness (RCS) of UHS is constantly changed with construction. In order to determine the RCS of UHS in situ, parametric analysis was performed by establishing a theoretical mechanical model of plane rotation and a refined solid finite element (FE) model of UHS. The slope of the linear rising segment of the load–displacement curve obtained from the numerical simulation was considered as the RCS of the UHS. The relationships between RCS and the vertical force, geometric parameters, and material parameters were established, and then the calculation formula of RCS was further proposed. To verify the accuracy of the proposed formula, a case study for a real bridge was conducted in this paper. The results show that the error of the tower rotation displacement can be reduced by about 90% using the proposed method compared with the conventional method, which regards the hinge as an ideal one, and the precision is greatly improved. This study has enormous potential to quickly determinate the RCS of UHS in practical applications, and plays a great promotion role in enriching the structural system of cable-stayed bridges.