A practical iterative tuning algorithm of a decoupling controller is presented to eliminate intrinsic coupling among 3-DOF precision motion stage. General decoupling control cannot eliminate coupling completely, which will prevent the enhancement of control accuracy in high-precision motion system. The proposed algorithm can be used to tune the parameters of a decoupling controller iteratively through minimizing a quadratic cost function of the tracking error in non-movement direction when the stage moves in one direction. The tuning algorithm addressed for the motion stage needs only measurement signals in an actual motion system rather than a detailed model of the stage. The proposed algorithm is demonstrated by experimental results. It can be applied further in other multi-DOF motion systems.