To investigate the vertical prestress loss in concrete box girder webs with twice-tension, a rectangular thin plate test under twice-tension prestressed strand was conducted. At the same time, twice-tension vertical prestress loss used in a practical bridge was also tested, and was compared with the traditional vertical prestressed system using finishing rolling rebar. The test results have confirmed that the immediate vertical prestress loss can be reduced to less than 10% with the twice-tension technology, far less than that of finishing rolling rebar system. Twice-tension system can improve the efficiency of vertical prestress and shear reliability in box girder webs. At last, based on the test results, some factors that affect vertical prestress loss, including pipe roughness, compression of joint, deformation and retraction of anchorage, elastic compression of concrete, relaxation of tendons and creep and shrinkage of concrete, were discussed, and the corresponding methods of design taking value are proposed.