Biosensors based on a field-effect transistor platform allow continuous monitoring of biologically active species with high sensitivity due to the amplification capability of detected signals. To date, a large number of sensors for biogenic substances have used high-cost enzyme immobilization methods. Here, highly sensitive organic field-effect transistor (OFET)-based sensors functionalized with synthetic receptors are reported that can selectively detect acetylcholine (ACh+), a critical ion related to the delivery of neural stimulation. A cucurbituril (CB) derivative, perallyloxyCB ((allyloxy)12CB, AOCB), which is soluble in methanol but insoluble in water, has been solution-deposited as a selective sensing layer onto a water-stable p-channel semiconductor, 5,5′-bis-(7-dodecyl-9H-fluoren-2-yl)-2,2′-bithiophene layer. The OFET-based sensors exhibit a detection limit down to 1 × 10–12 m of ACh+, which is six orders of magnitude lower than that of ion-selective electrode-based sensors. Moreover, these OFET-based sensors show highly selective discrimination of ACh+ over choline (Ch+). The findings demonstrate a viable method for the fabrication of OFET-based biosensors with high sensitivity and selectivity, and allow for practical applications of OFETs as high-performance sensors for biogenic substances.