The temperature effect of new-type composite box girder with corrugated steel webs (CSWs) is prominent due to the significant difference of thermal parameters between concrete and steel. A theoretical calculation method for relative slip, internal force and stress of new composite box girder with CSWs under vertical temperature gradient was established. The equilibrium condition of sub-girder, deformation coordination condition between sub-girders and shear deformation effect of CSWs were considered. The temperature of new composite box test beam with CSWs in large temperature difference area was observed for a long time, and the vertical temperature gradient function of the structure was fitted. The temperature response of the structure under the measured temperature gradient was calculated by the theoretical method, and the theory was verified by finite element simulation. Results show that the interfacial shear force, the bending moment and the stress of the beam are all distributed as hyperbolic cosine function along the longitudinal direction of the beam under the measured temperature gradient. The relative slip between layers is distributed as hyperbolic sine function along the longitudinal direction of the beam. Whether the shear deformation effect of webs is considered greatly influences on the temperature effect in the range of 0.8 m from the end to the middle of the composite beam, and the effect on the middle of the composite beam can be ignored. The linear expansion coefficient of concrete, the sliding stiffness between layers and the interface temperature difference of composite box girder greatly influence on the temperature effect of the new composite box girder with CSWs. The interlayer shear connectors should be reasonably arranged in the design, and the temperature effect of the new composite box girder with CSWs should be calculated by considering the variation of the linear expansion coefficient of concrete.