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In advanced industrial automation, industrial robots have been widely utilized on assembly lines in order to reduce labor dependence. However, many related layout design approaches proposed are prone to generating unsafe layouts: there generally lacks a consideration regarding robots’ heights and assembly range, which will lead to costly collisions in the operation stage. In order to address the problem, we propose a three-dimensional (3D) optimization approach to a safe layout design for an assembly line with robots. We define modeling rules for robots to judge assembly ranges. A quantitative safety indicator is employed as a trigger for 3D collision detection in order to determine the positional relationship and status of the safe assembly collaboration. The optimization goals are established for minimizing the logistical cost and layout area in the model. A combined algorithm of differential evolution and nondominated sequencing genetic II is applied, which effectively enhances the poor diversity and convergence of the mainstream optimization method when solving this model. The benchmark tests and validation proved that our approach yields excellent convergence and distribution performance. The case study verifies that the safe layout model is valid and our approach can generate a safe layout in order to optimize economics and safety.