Transfer reaction characteristics of tetracycline (TC) across a polarized water/1,2-dichloroethane (1,2-DCE) interface was studied via controlling both pH and ionic strength of the aqueous phase in conjunction with cyclic and differential pulse voltammetries. Formal transfer potential values of differently charged TC ionic species at the water/1,2-DCE interface were measured as a function of pH values of the aqueous solution, which led to establishing an ionic partition diagram for TC. As a result, we could identify which TC ionic species are more dominant in the aqueous or organic phase. Thermodynamic properties including the formal transfer potential, partition coefficient and Gibbs transfer energy of TC ionic species at the water/1,2-DCE interface were also estimated. In order to develop an electrochemical sensor for TC, a single microhole supported water/polyvinylchloride- 2-nitrophenyloctylether (PVC-NPOE) gel interface was fabricated. A well-defined voltammetric response at pH 4.0 associated with the TC ion transfer process was achieved similar to that of using the water/1,2-DCE interface. Also the measured current increased proportionally with respect to the TC concentration. A 5 μM of TC in pH 4.0 buffer solution with a dynamic range from 5 μM to 30 μM TC concentration could be detected when using differential pulse stripping voltammetry.