The invention discloses a fatigue testing method for a rail transportation vehicle body welding structure, and belongs to the technical field of fatigue testing. The method comprises the following steps: 1, making preparations before testing: performing numerical simulation analysis to determine a load value; 2, assembling a test sample and a fixture: rigidly clamping and fixing a straight vehicle body section of the vehicle body welding structure test sample by adopting a rigid clamping end and elastically clamping and fixing an irregularly-shaped vehicle body section by adopting an elastic clamping end, to form a combined test body; 3, fixing the combined test body on a fatigue tester; 4, performing loading test according to the load value; 5, performing detection. According to the method, any shape can be effectively fixed and clamped by rigid and flexible combination, and the actual stress centerline of the welding structure is overlapped with the loading stress axis of the fatigue tester, so that the fatigue level of the rail transportation vehicle body welding structure can be accurately measured.