Machine tool joint stiffness has important affects on the dynamic characteristics of the whole machine tool. It is a challenging task to optimize multi-joint stiffness of the overall structure. By discussing the distribution of stiffness of linear guides, ball screws, bolt joints and bearings, a discrete optimization method of multi-joint stiffness of a machining center was presented based on orthogonal experiment and FEA. Mean frequency formulation was adopted to define the index of orthogonal optimization. According to the principle of orthogonal experiment, optimization levels were found out, sequence of the factors on the index of the orthogonal experiment was listed and significant factors were determined. Results of this research proved the validity and feasibility of the discrete optimization method of multi-joint stiffness of the whole machine tool by combining orthogonal experiment and FEA.