To study the fatigue performance of a composite girder with corrugated steel web (CSW) and steel tube (ST) truss, based on an engineering object, a test and finite element (FE) analysis were carried out. The fatigue failure mode and the sensitivities of the hot spot stress to the geometric parameters of the composite girder above were studied. The position with the maximum hot spot stress and the fatigue crack position were discussed and determined. Furthermore, a fatigue life evaluation method was discussed. It was found that linear extrapolation was applicable to finding the hot spot stress. The fatigue performance evolution comprised three stages, including initiation, expansion, and failure of the fatigue crack. The fatigue crack initiation stage accounted for about 95.6% of its fatigue life, and the fatigue crack was opening type I. The maximum hot spot stress emerged at the endpoint of the inclined web of CSW (point S). The main reason for the behavior was that the inclined web had a small out-of-plane flexural stiffness but had to bear an out-of-plane bending moment. Moreover, for the same section of the CSW, the hot spot stress on the molding side was less than that on the non-molding side. The hot spot stress exhibited negative correlations with the chord diameter, chord thickness, and bend radius, meanwhile, it had the highest sensitivity to the chord diameter. The existing S-N curves were not suitable to evaluate the fatigue life of the composite girder with CSW and ST truss. Since the intersecting weld between the CSW and the chord was located in longitudinal welded joints, the hot spot stress method suggested adopting the basic fatigue strength of 100 MPa, corresponding to 2,000,000 load cycles. Meanwhile, an empirical calculation model of the S-N curve for the composite girder with CSW and ST truss was created.