Three-phase hollow fiber based liquid phase microextraction (HF-LPME) was developed for the preconcentration of kanamycin sulfate combined with electrochemiluminescence detection. The extractant was 1-octyl-methylmidazolium hexafluorophosphate ([OMIM]PF6). A hollow fiber supported liquid membrane was used between phosphate buffer solution (PBS) containing analyte (kanamycin sulfate) as donor phase and aqueous solution (pH 10) as acceptor phase. The effects of various parameters on the extraction efficiency were studied, such as pH of ECL working solution, extraction temperature, pH of donor phase, extraction time and salt content of sample. Under the optimized conditions, the linear range was 0.002-0.1 mu g mL(-1), and the limit of detection (LOD) was 0.00067 mu g mL(-1). No expensive equipment is necessary to perform this assay which makes it a viable replacement. (C) 2014 Elsevier B.V. All rights reserved.