To study the influence of interlayer stiffness and interlayer damping on the dynamic response of multi-layer beams under moving loads, a method for calculating the dynamic response of a multi-layer beam system considering random interlayer stiffness or random interlayer damping under successive moving loads based on the Karhunen–Loéve expansion and point estimation method is proposed. The accuracy of the proposed method was verified by using several numerical examples. The influence of interlayer stiffness, interlayer damping, standard deviation of interlayer stiffness, and standard deviation of interlayer damping on the dynamic response of a girder-rail system was analyzed by using the proposed calculation method. The results show that when the moving load velocity is high, the number of critical velocity of the rail significantly increases with decreasing interlayer stiffness. In addition, dynamic responses of the rail and girder increase with decreasing interlayer damping and the dynamic response of rail at the midspan significantly increases as the standard deviation of interlayer stiffness increases. Neglecting interlayer damping can lead to significant fluctuation in the dynamic response of rail, a significant increase in the number of critical velocity of rail, and increases in the dynamic responses of rail and girder. Changing interlayer stiffness, interlayer damping, standard deviation of interlayer stiffness, or standard deviation of interlayer damping will have no obvious influence on the dynamic response of girder.