The transfer process of an ionizable fluoroquinolone antibacterial drug, levofloxacin (LVX), at a polarized interface between two immiscible electrolytes solutions (ITIES) has been investigated for the first time using voltammetric techniques. The charged nature of ionizable LVX species at various aqueous pH solutions with controlled ionic strength were characterized and further used for establishing an ionic partition diagram of LVX. The results led us to predict the dominant charge species with respect to the pH of aqueous solutions and LVX lipophilicity in addition to the thermodynamics information of the LVX transfer process. A voltammetric sensor featuring a microhole supported water/organic gel interface, based on measuring current changes associated with the protonated LVX (HLVXthorn) transfer reaction across the interface, was also developed. Our HLVXthorn sensitive sensor in conjunction with a differential pulse stripping voltammetry (DPSV) technique was employed for the quantification of HLVXthorn in buffer solution and further for food sample via spiking HLVXthorn in a commercially available milk solution. Our sensing results were finally validated with those using high-performance liquid chromatography (HPLC) analyses. (C) 2018 Elsevier Ltd. All rights reserved.