Objective In response to the clinical needs for personalized wrist orthoses, a topological optimization design method was proposed to achieve an integrated macro- and micro-structural optimization of a personalized, lightweight, and comfortable wrist orthosis. Methods A composite biomechanical finite element model of the wrist orthosis and upper limb was established to quantify the effects of the orthosis geometry on its fixation performance and comfort. A multi-condition topological optimization and microstructure design approach was employed to optimize the non-load-bearing areas of the orthosis. The orthosis was manufactured using three-dimensional (3D)-printed polyether ether ketone (PEEK), and the feasibility of the design was validated. Results While maintaining mechanical strength, the weight of the 3D-printed PEEK orthosis was reduced by 28% compared to the traditional orthoses. Both the pressure at the skin contact interface and the results of a subjective questionnaire indicated that test subjects experienced a high level of comfort wearing the orthosis. Conclusions The orthosis design achieved personalization, lightweight structure, and high comfort while ensuring mechanical strength and fixation performance.