Ion-sensitive transistors with nanoscale or microscale dimensions are promising for high-resolution electrophysiological recording and sensing. Technology that can pattern polymer functional materials directly from a solution can effectively avoid any chemical damage induced by conventional lithography techniques. The application of a mold-guided drying technique to pattern PEDOT:PSS-based transistors with high resolution directly from the water-based suspension is presented in this paper. Gold electrodes with short channels were first defined by creating high-resolution polymer lines with mold-guided drying followed by pattern transfer through a lift-off process. Then, PEDOT:PSS lines were subsequently created through an identical mold-guided drying process on the predefined electrodes. Small-scale transistor devices with both shortened channel length and width exhibited a good high-frequency response because of the weak capacitive effect. This is particularly advantageous for electrochemical transistors since the use of conventional fabrication techniques is extremely challenging in this case. In addition, modified polymer chain alignment of the assembled PEDOT:PSS lines during the drying process was observed by optical and electrical measurement. The mold-guided drying technique has been proven to be a promising method to fabricate small-scale devices, especially for biological applications.