Tetraethylammonium (TEA) is a common molecular probe in detecting potassium channel blocking. The external TEA binding affinity has been proposed to be highly related to the aromatic residue located at the outer mouth of the potassium channels such as Y82 in KcsA and Y449 in Shaker, probably due to the π-cation interaction between TEA and the aromatic side chain. In this report, we identified the highly sensitive TEA block for the chlorella virus-encoded Kcv, a miniature model K+ channel that only consists of 94 amino acids with two transmembrane domains and a conservative selectivity filter. By mutagenesis screening at Leu70 of Kcv, which is equivalent to the TEA site Y82 in KcsA, we found substitution of Leu70 to all other amino acids including Tyr, Phe and His will reduce the TEA affinity, suggesting a more complicated mechanism beyond cation-π interaction involved in TEA blocking. We further developed a novel functional stoichiometric approach to exploring how each individual subunit contributes to the TEA binding. We co-expressed the mutant Kcv and a mass-tagged wild-type Kcv, to form hetero-tetramers that can be electrophoretically separated. Because Kcv is able to retain the channel-forming function in detergent SDS [FEBS Lett. 581, 1027-1034 (2007)], we can purify all types of hetero-channels directly from the SDS gel, and subject to single channel recording. Through this approach, we established a linear correlation between the free energy for TEA blocking and the number of mutant subunits in a tetramer, which infers that each subunit independently interacts with one ethyl group of TEA and contributes equal energy to the overall TEA affinity. The functional stoichiometric approach we developed with purified hetero-channels can be applied to the mechanism study of many K+ channel drugs and inhibitors.