Charge trapping is a long-standing problem in electrowetting on dielectric, causing reliability reduction and restricting its practical applications. Although this phenomenon is investigated macroscopically, the microscopic investigations are still lacking. In this work, the trapped charges are proven to be localized at the three-phase contact line (TPCL) region by using three detecting methods—local contact angle measurements, electrowetting (EW) probe, and Kelvin probe force microscopy. Moreover, it is demonstrated that this EW-assisted charge injection (EWCI) process can be utilized as a simple and low-cost method to deposit charges on fluoropolymer surfaces. Charge densities near the TPCL up to 0.46 mC m−2 and line widths of the deposited charge ranging from 20 to 300 µm are achieved by the proposed EWCI method. Particularly, negative charge densities do not degrade even after a “harsh” testing with a water droplet on top of the sample surfaces for 12 h, as well as after being treated by water vapor for 3 h. These findings provide an approach for applications which desire stable and controllable surface charges. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.