This work reported the construction of a chiral sensor based on an organic field-effect transistor to probe the subtle change of weak interactions in the chiral discrimination process, with the ability to achieve fast, sensitive, and quantitative real-time chiral analysis for various racemic pairs, where a β-cyclodextrin (β-CD) sensitized copper hexadecafluorophthalocyanine (F16CuPc) semiconductive layer was employed as the sensing unit. Physical adsorptive assembly of β-CD on the semiconductive layer guarantees the impressive field-effect-amplified chiral sensitivity. The enantiomer induced aggregation pattern diversification of the sensing layer resulted in enhanced or weakened surface-dipole interactions to various degrees and hence brought about the drain current fluctuation. A fast and real-time detection of the enantiomer pairs in aqueous solution at 10-9 M was achieved. This chiral organic field-effect transistor (COFET) afforded reliable ability for quantitative determination of the pure isomer content in enantiomer pairs and was further proven to have great potential for the resolution of "real-world" pharmaceutical drugs.