The field of liquid crystal display (LCD) is constantly in the spotlight and the process of depositing an alignment layer in the LCD manufacturing process is very important to obtain excellent performance such as low-power driving and high-speed response to improve LCD performance. Therefore, research on liquid crystal (LC) alignment is being actively conducted. When manufacturing LCD, it is necessary to consider the effect of the alignment layer thickness as one of the factors affecting various LCD performances. In addition, previous studies confirmed the LC alignment characteristics correlate with the rotation speed in the spin coating process. Therefore, the electro-optical properties of the LCD were investigated by manufacturing a polyimide alignment layer by varying the rotation speed in the spin coating process in this study. It was confirmed that the thickness of the polyimide alignment layer was controlled according to the spin coating conditions. The average transmittances of anti-parallel LC cells at the spin coating speed of 2,500 rpm and 3,000 rpm are about 60%, which indicates that the LC cell has relatively higher performance. At the spin coating speed of 3,000 rpm, the voltage-transmittance curve of twisted nematic (TN) LC cell was below 1.5 V, which means that the TN LC cell operated at a low power. In addition, high-speed operating characteristics were confirmed with a response time of less than 30 ms. From these derived data, we confirmed that the ideal spin coating speed is 3,000 rpm. And these results provide an optimized polyimide alignment layer process when considering enhanced future LCD manufacturing.