The impedance analysis of single cells can provide accurate and in-depth information about their pathological condition. However, the electrical properties of single cells will be affected by measurement environment such as cell location, electrodes geometry and so on. Accordingly, this study presents the relationship between impedance characteristics and location of the single HeLa cell. This study uses a microfluidic device to capture physically single cells with three-pillars microstructure and measure the impedance of a single HeLa cell (human cervical epithelioid carcinoma) using impedance spectroscopy. Additionally, in order to understand the relationship between impedance characteristics and location of the single HeLa cell, the equivalent circuit model is established and an element.The measurement results show that the magnitude of a single HeLa cell impedance decreases and the phase of the single HeLa cell impedance changes from the hook line to the inverse line with the operating frequency from 1 to 100 kHz at operating voltage of 0.7 V in the time from 1 to 19 hours because the distance between the single HeLa cell and electrodes decrease. The single cell falls toward electrodes as time elapses after the single cell is trapped by three-pillars microstructure.