Ballastless track is widely used in high-speed railways due to its long service life, low maintenance costs, and excellent riding comfort. However, laying a ballastless track on a long-span bridge remains a challenge because of the large deflection and uneven stiffness of the long-span bridge. Limited research has been reported on the experimental behavior of the long-span railway bridge laid with the ballastless track. This study presents the in-situ testing and the finite element model updating (FEMU) of the Ganjiang Bridge, China’s first long-span ballastless railway cable-stayed bridge with a main span that reaches up to 300 m before its opening for operation. The study’s primary purposes include evaluating the mechanical properties of the ballastless railway bridge and establishing a baseline finite element model for subsequent structural health monitoring. The general test plan, sensor placement, and measured responses are described. The static deflection, the stress of the main girder, and the rotation angle at the beam end are used to evaluate the static performance of the bridge. The natural frequencies and mode shapes are used to assess the dynamic performance of the bridge. A FEMU framework that utilizes displacement assurance criterion (DAC) and frequency to construct a single-objective function is proposed to update the initial finite element (FE) model of the bridge. The results show that the deflection to span ratio is 1/1016 (less than1/1000), and the rotation angle at the beam end under live load is 0.032 ‰ rad (<1‰), indicating that the bridge has sufficient vertical stiffness and excellent riding comfort. The stress of the main girder is smaller than the material yield strength, indicating that the bridge has sufficient load-bearing capacity. All the requirements allowing the bridge to start service have been met. The updated FE model shows that the DAC performs better than the displacement and provides more accurate updating results. The updated FE model can well reflect the static and dynamic characteristics of the actual bridge and can be used as a baseline FE model for subsequent studies.