In order to investigate the feasibility of the virtual static load test method for evaluating the stiffness of the main girder of a large-span cable-stayed bridge, a cable-stayed bridge was subjected to a static load test and a modal test under environmental excitation. The static deflection of the measuring points at the mid-span section of the main girder was measured in the static load test. Modal expansion of the measured mode shapes of the cable-stayed bridge was carried out, and the Kriging interpolation method was applied to predict the modal deflection of the mid-span section of the cable-stayed bridge under the static load test. A virtual static load test scheme meeting the loading efficiency requirement was designed, and the deflection calibration coefficient under virtual static load was calculated to evaluate the stiffness of the main girder of the cable-stayed bridge. The results show that the modal test under ambient excitation can accurately obtain the bridge vibration information; the relative error between the modal deflection predicted by the Kriging interpolation method using only the first four orders of vertical modal parameters and the measured static load deflection is less than 10%, which meets the engineering accuracy requirement; the deflection calibration coefficient obtained by the virtual static load test method is close to that from the static load test, and the evaluation results are less than 1.0, which indicates that the main girder of the cable-stayed bridge is in good bearing condition and verifies the feasibility of the virtual static load test method for evaluating the stiffness of the main girder of a large-span cable-stayed bridge under normal traffic conditions.