Conductive polymer poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) exhibits a tunable conductivity ranging from 0.1 to 4380 S·cm–1 under different doping and/or dedoping strategies. However, the dependence of macroscopic electrical properties on the evolution of the microstructure is not clearly understood. This is the first study that systematically investigated the spatial arrangement of the ordered and disordered phases in PEDOT/PSS nanofilms by bright-field (BF), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) combined with electron energy loss spectroscopy (EELS) and element-thickness mapping. Our observations clarify how amorphous PSS hinders electrical transport at various length scales in the PEDOT/PSS films. Moreover, the mechanism for an enhancement in 3 orders of magnitude in electrical conductivity was proved by TEM investigation, which is mainly due to a more uniform dispersion by dedoping that opens PEDOT nanoparticle clusters in PEDOT/PSS films. Our microstructural and electrical studies show that the change in spatial arrangement and interaction of small PEDOT domains plays a considerable role in the final electron transport.