We aimed to characterize aging of single vascular endothelial cells, which are indicators of senescence, using micro electrical impedance spectroscopy (μEIS) for the first time. The proposed μEIS was equipped with two barriers under the membrane actuator near the sensing electrodes, increasing its cell-trapping capability and minimizing the interference between the target cell and subsequent cells. The cell-trapping capability in μEIS with barriers was considerably improved (90%) with a capture time of 5 s or less, compared to μEIS without barriers (30%). Cells were extracted from transgenic zebrafish to minimize an initial discrepancy originating from genetic differences. In order to estimate useful parameters, cytoplasm resistance and membrane capacitance were estimated by fitting an electrical equivalent circuit to the data of ex vivo sensor output. The estimated cytoplasm resistance and membrane capacitance in the younger vascular endothelial cells were 20.16 ± 0.79 kΩ and 17.46 ± 0.76 pF, respectively, whereas those in the older cells were 17.81 ± 0.98 kΩ and 20.08 ± 1.38 pF, respectively. Discrimination of each group with different aging showed statistical significance in terms of cytoplasm resistance (p < 0.001) and membrane capacitance (p < 0.001). Considering both of the sensor and cellular level, the optimal frequency was determined as 1 MHz at which the electrical impedance of each group was clearly discriminated (p < 0.001).