At present，there is no bridge design code to guide the fatigue design of beam with corrugated steel webs. In order to study the fatigue design and evaluation methods of this structure, constant amplitude fatigue load tests of 7 beams were performed, and the basic fatigue data were obtained. Based on the FEM sub model method and the linear extrapolation method recommended by the IIW, the hot spot stress concentration factor of model beams was calculated. The fatigue performance of tested beams in this paper and other two tests was estimated by the hot spot stress method. The results show that the hot spot stress method is safe in evaluating the fatigue test results of the beams with corrugated steel webs and the 100 FAT class is recommended for fillet welds in this structure. The parameter analysis indicates that the largest hot spot stress and its concentration factor is greatly influenced by steel flange thickness and web corrugation angle, and they decrease with the increment of steel flange thickness and increase with the increment of web corrugation angle, while they are less affected by the web corner radius and decrease with the increment of web corner radius.