Single-row layout is widely used within GT cells, in facilities that implement JIT, and sometimes with FMS. The three types of single-row layout, including linear, U-shape, and semi-circular layout are introduced firstly. Based on the lower selling price of customers’ demand, the components of manufacturing cost are analyzed secondly. Then the relationship between the manufacturing cost and objectives of single-row layout are described. Meanwhile, the models related to several parts of manufacturing cost of single-row layout are set up respectively. Finally, the simulation of these models for three types of single-row layout is running in QUEST. After analyzing the simulation results, conclusions are obtained: (1) area and labor utilization of linear layout are higher than the others, (2) material handling cost of semi-circular is lowest among these three types of single-row layout, and (3) equipment utilization of them are almost the same. This research results will provide the references in selecting the types of single-row layout based on manufacturing cost.