INTRODUCTION Nicotine, a highly addictive substance, is naturally produced in the Solanaceae family of plants, particularly tobacco. The presence of nicotine in plant foods has adverse effects on the lungs, kidneys, heart, and reproductive system. OBJECTIVE A novel three-phase microchip flat electromembrane coupled with online high-performance liquid chromatography (HPLC) was developed to analyze nicotine in tomato, mushroom, eggplant, bell pepper, and red pepper. METHODS The microchip was connected to the HPLC in online mode. All effective variables were optimized to achieve the best extraction response. The use of electric potential and 2-nitrophenyl octyl ether -5% di(2-ethylhexyl) phosphate as a modified supported liquid membrane (SLM) increased the sensitivity and selectivity. RESULTS The optimal extraction voltage, extraction time, and ion balance were 40 V, 10 min and 0, respectively. The dynamic linear range was 0.5-1000 ng g-1. The obtained recovery, relative standard deviation, and enrichment factor were 98%, 7%, and 35, respectively. The limits of detection 0.4 ng g-1 and the limits of quantification were obtained 1.3 ng g-1. The highest (105.0 ng g-1) and lowest (3.4 ng g-1) concentrations of nicotine were obtained for eggplant and tomato, respectively. CONCLUSION Selective electromembrane extraction of nicotine from the donor phase to the acceptor phase was performed by optimizing the main variables influencing the method mechanism. The new channel design in this analytical system and online injection increased efficiency, stability, and repeatability. The results revealed that this method is capable for the efficient determination of trace amount of nicotine in edible vegetables.