The development of 3D Cell-printing technology contributes to the application of tissue constructs in vitro in neuroscience. Collecting neural cells from patients is an efficient way of monitoring health of an individual target, which, in turn, benefits the enhancement of medicines. The fabricated sample of neural cells is exposed to potential drugs for the analysis of neuron responses. 3D cell-printing as an emerging biofabrication technology has been widely used to mimic natural 3D models in in vitro tissue research, especially in vitro brain-like tissue constructs in neuroscience. Fabricated brain-like tissue constructs provide a 3D microenvironment for primary neural cells to grow within. After more than several weeks of in vitro culturing, the formation of neural circuits in structures equips them with the capability of sensitively responding to a stimulus. In this study, an in vitro layered brain-like tissue construct is first proposed and later developed by 3D cell-printing technology. The layered structure is systematically analyzed, starting from printing parameter optimization to biological functionality performance. The optimized diameter of printing nozzle and printing speed are 0.51 mm and 5 μl s-1, respectively, and the elastic modulus is approximately 6 kPa. Live/dead and immunostaining imaging is used to verify the growth of neural cells in the printed structure. The survival rate of neural cells in 2D and 3D samples is compared, and the results demonstrate that the 3D-printed structures exhibit a better artificial culturing environment and a higher survival rate. Both 2D and 3D samples are directly cultured in a 4 × 4 multiple electrode array. Local field potentials are collected and validated by the Med64 recording system. Tetrodotoxin is used to test the drug sensitivity of the printed structure, and the excitatory postsynaptic potential signals of the physiological performance indicate that the 3D-printed structure has great potential as a drug testing model in the pharmaypeceutical study.