The poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) film is a promising material for electrodes, biomolecular sensor channels, and probes for physiological signals because the electrical conduction of PEDOT:PSS is tuned simply through the electrochemical reaction with the target analyte. However, forming a specific morphology or nanostructure on PEDOT:PSS thin films immersed in an aqueous solution is still a challenge. Herein, we report the mechanism for the stepwise morphological change in the highly conductive PEDOT:PSS layer that successfully explains the electrical and structural modulations that occur after a soaking test in various pH conditions. The change in PEDOT:PSS begins with the rapid swelling and dissolution of PSS-rich domains and the simultaneous structural rearrangement of the remaining PEDOT chains within 1 s of dipping. Analysis confirms that the pH conditions of an aqueous solution govern the oxidation state and the form of the PEDOT chains. After removing the water molecules, additional PEDOT-rich grains were generated and accumulated on the surface of the film, which exhibited hydrophobic barrier characteristics. With the help of this intrinsic barrier on the PEDOT:PSS surface, the sheet resistance slightly increased from 72 to 144 Ω/sq even after dipping in a water bath for 350 h. We also demonstrate the usability of the proposed approach on a sensor to detect vitamin C in an aqueous medium. Utilizing the electrochemical reaction of PEDOT:PSS films, the simple resistor sensor showed a response time of less than 150 s, which is 10 times faster than that observed in a previous report. The soaked samples also showed a more reliable linear correlation between the current change and the amount of ascorbic acid compared with pristine PEDOT:PSS. Both the proposed mechanism and the role of accumulated PEDOT-rich regions illustrate the versatile potential of highly conductive PEDOT:PSS films in the field of bioelectronic applications, owing to the increased design architecture.