In the present work, the micellar electrophoretic separation of five chlorophenols (CPs) on a functionalized poly(dimethylsiloxane) (PDMS) microchip with amperometric detection was performed. In order to achieve high resolution by controlling the electroosmotic flow (EOF) as well as signal detection by suppressing analytes adsorption, the microchannel was functionalized by poly(diallyldimethylammonium chloride) (PDDA)/SiO2 nanospheres (NSs)/ poly(sodium-p-styrenesulfonate) (PSS), via an approach of layer-by-layer assembly. Five chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,3-dichlorophenol and 2,4,6-trichlorophenol) were efficiently separated in this microchannel (3.7 cm of length) within 120 s. A resolution of at least 2.4 was obtained with a 10 mM phosphate buffer solution (PBS) (pH 9.48) containing 20 mM sodium dodecyl sulfate (SDS) and 50% (v/v) acetonitrile as a carrier under optimized conditions. A graphene-modified carbon microdisk electrode was used for high-sensitivity detection. Its characteristics were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). For those CPs, linear ranges of 0.08-5, 0.06-5, 0.04-5, 0.04-5 and 0.30-20 μM and detection limits of 0.021, 0.026, 0.022, 0.019 and 0.054 μM were obtained, respectively. The method was successfully applied for the analysis of some wastewater samples with satisfactory recovery.