Among various health care demands for ageing population, the total knee arthroplasty (TKA) is currently one of the major health care expenses. TKA is a process to replace the surfaces of femur and tibia with the knee prosthesis, i.e., femoral component, tibial tray, and spacer. During service, the metal tibial tray is under cyclic bending; thus, the fatigue failure is possible. The cyclic bending and fatigue failure of tibial tray can be enhanced by the malalignment of knee prosthesis due to the change of bodily structure in ageing population. To investigate the influence of malaligned knee prosthesis on fatigue life, the numerical stress analysis and in-vitro fatigue test of tibial tray based on ASTM F1800 were performed. For tibial trays with malalignments, the location of the maximum stress range was near the junction between stem and tray, which corresponded to the location of fractures observed previously in clinical application.