In order to study the characteristics of unsteady seepage in the filling and emptying process of lock chamber with a non-homogeneous layered ground, according to the theory of basis seepage flow, this study divides the seepage into the consolidation seepage and the instantaneous steady seepage. Based on the finite element software ABAQUS, the two-dimensional seepage model has been built according to a project example, in order to simulate the filling/emptying process of lock chamber considering fluid-solid coupling and analyze the characteristics and effect of unsteady seepage. The result shows that, with the cyclical variations of filling and emptying, the piezometric level has three characteristics: cyclicity, decay and hysteresis. In the filling and emptying process, zero velocity points exist. In addition, in the end of emptying process, the infiltration stress of unsteady seepage is greater than that of steady seepage with a low water level, the stability coefficient decreases and the possibility of seepage failure grows. Therefore, the influence of unsteady seepage field should be taken into account in the anti-seepage design of ship lock.