In order to explore a new type of composite box girder with light lifting weight, convenient construction and high durability, a new type of prestressed RC-UHPC composite box girder with ultra-high performance concrete (UHPC) as web and bottom plate and plain concrete (RC) as top plate was designed and fabricated. Flexural tests and finite element analysis were carried out to investigate the failure mechanism and failure mode of the specimens. The results show that: the failure mode of the specimen is suitable reinforcement failure, which shows that the RC of the top slab in the pure bending section is crushed after the yield of the longitudinal tensile reinforcement and the fracture of some prestressing reinforcement; the tensile strength of UHPC and the reinforcement ratio of prestressing tendons have a greater influence on the flexural strength; the longitudinal strain of the web of the specimen is basically consistent with the assumption of a flat section in the height direction. Combined with the theoretical analysis, the formulas for calculating the flexural strength were proposed. The ratio of the calculated value to the test value and the calculated value of the finite element is 1.014 and 0.960, respectively, which has a high calculation accuracy. This study can provide a theoretical basis for the promotion of prestressed RC-UHPC composite box girder in practical projects and the formulation of specifications.