In this study, based on ion-current rectification in the conically shaped nanochannel embedded in polyethylene terephthalate (PET) membrane, we have selectively discriminated three small biomolecules. Because three positive biomolecules (Hoechst 33342, Propidium and Bupivacaine) have different hydrophobicities, their interactions with inside wall of the conical nanochannel are different and their binding affinities can be derived from Langmuir absorption model. Therefore, we can successfully discriminate these small biomolecules. The highest binding constant was obtained for the small molecule with highest hydrophobicity. Another interesting result is that the detection limit for the small molecule with the highest binding constant shifts to submicromole.Highlights► Hydrophobic biomolecules can be selectively detected via nanopore. ► The detection limit for each one is dependent on its affinity toward the membrane. ► The molecule with the highest binding affinity has the lowest detection limit.