Cell culture is the basis of cell research. In order to provide an approximate microenvironment for cell culture, a micro-device for three-dimensional and dynamic cell culture was designed. First, a micro-channel network was designed for transportation of liquid. The cell culture chamber was symmetrically arranged in the micro-channel network. A series of “multiple input and multiple output” micro-channels were connected with the inlet and outlet of the cell culture chamber. Second, the physical fields of laminar and porous media in the COMSOL software were coupled, which was used to simulate the velocity of the liquid in the cell culture chamber of the micro-device. The network structure of micro-channels was optimized by comparing the homogeneity and stability of the flow field. Third, the electrohydrodynamic direct-writing technology was used to integrate polycaprolactone (PCL) three-dimensional scaffolds in the cell culture chamber, which constructed the three-dimensional culture space. Finally, after the welding of the micro-device, the fluid flow condition in the cell culture chamber of the micro-device was tested, and cell experiments were performed. The results show that the fluid stability and homogeneity in the cell culture chamber of a “2×2” micro-device are appropriate. The fiber spacing of the three-dimensional scaffolds is 400 μm, the diameter is 80 μm, and the porosity is 64%. Besides, the cell survival rate is more than 90%. The micro-device, which can be used for three-dimensional dynamic cell culture, precisely simulates the microenvironment needed for the survival of cells in vivo. Cells in the culturing chamber grew well; therefore, the micro-device can satisfy the design requirements.