In order to improve the processing efficiency, the motion planning and path optimization of a three-axis Cartesian coordinate manipulator, which is designed for the drilling of holes in piano chord axis plates, are investigated. In this investigation, the linear interpolation algorithm with parabolic transition is applied to the motion planning of the drilling, and a smooth trajectory with continuous position and velocity is generated. Then, a mathematical model describing the path planning for holes machining is established, with the shortest processing path as the object and with a collision-free way to bypass all the obstacles as the constraint. Moreover, a multi-layer genetic algorithm is used to find the solution to the mathematical model. Finally, six types of piano chord axis plates are used for algorithm verification. The results verify the effectiveness of the proposed algorithm.