In order to explore a new type of composite box girder with light hosting weight, convenient construction and high durability, a new prestressed RC-UHPC composite box girder with ultra-high performance concrete (UHPC) as web and bottom plate and reinforced concrete (RC) as top plate was designed and fabricated. A flexural test was carried out to investigate the failure mechanism and failure mode of the specimens; the finite element software was utilized to simulate the test, and the calculation accuracy of the finite element method was verified by comparing the finite element calculation results with the test data, on the basis of which 16 finite element analysis models were established to analyse the influence of various parameters on the flexural strength. The results show that the failure mode of the specimen is adequate reinforcement failure, which shows that the RC top slab in the pure bending section is crushed after the yielding of the longitudinal tensile reinforcement and the fracture of some prestressing reinforcement; the tensile strength of UHPC and the reinforcement ratio of prestressing tendons exert a greater influence on the flexural strength; the longitudinal strain of the specimen’s is basically consistent with the plane section assumption in the height direction. Combined with the theoretical analysis, the formulas for calculating the flexural strength were proposed. The ratios of the calculated value to the test value and the finite element calculated value are 1.014 and 0.960, respectively, which indicates a high calculation accuracy.