When a railway project passes through an ecologically sensitive area, the reasonable alignment scheme should not only save the investment but also protect the ecological environment. There are various special structure design and construction approaches in a railway project for ecological protection which can be impressed by cost items. However, it is inevitable to acquire some land for the permanent structures of bridge, subgrade, tunnel, and station, where the primitive vegetation and soil are destroyed or disturbed. This chapter proposes a bi-objective optimization model for railway alignment considering the descent of investment and ecological damage simultaneously. Firstly, the structure design and construction approaches for ecological protection are discussed. The principles to set the investment objective considering ecological protection are proposed; Secondly, the ecological objective function to evaluate the ecological damage is described. Thirdly, the bi-level and bi-objective Optimization model of railway alignment is proposed and a multi-objective optimization based on a DE algorithm and decomposition strategy is introduced to solve the optimization model. The case study shows that the suggested optimization model can generate a set of diverse horizontal alignments most of which are on a Pareto Front.