This paper reports on the study of electroosmotic flow (EOF) in poly(dimethylsiloxane) (PDMS) microchannels on the basis of indirect amperometric detection method. Gradual increase of EOF rate in freshly prepared PDMS microchannels was observed with the running buffer of phosphate buffer solution (PBS). With the same concentration (10 mM) of PBS containing different cations and the same pH value (7.0) and, the time of the stable EOF in PDMS microchannels under the applied separation voltage of 1000 V was 49.8 s (Li+ -PBS), 57.1 s (Na+ -PBS), 91 s (K+ -PBS), respectively. Meanwhile, the different adsorption of cations (Li+, Na+ and K+) on hydrophobic PDMS wall was observed through their separation in PDMS microchannels. Such experimental results demonstrated that the EOF in PDMS microchannels came from the cations and anions adsorbed on PDMS wall. This study would not only help us understand the surface state of PDMS, but also provide a useful guidance for establishing the effective surface modification methods in PDMS microchip CE.