Separation and direct detection of amino acids, glucose and peptide in a 3.1 cm separation channel made of poly(dimethylsiloxane) (PDMS) with end-column amperometric detection at a copper microdisk electrode was developed. This system is the integration of a normal sized working electrode with electrochemical detection on a PDMS microfabricated device. The PDMS channels dynamically modified by 2-morpholinoethanesulfonic acid (MES) show less adsorption and more enhanced efficiency than that of unmodified ones when applied to separations of these biological molecules. The migration time is less than 100 s and the reproducibility of migration time is satisfactory with relative standard deviation (RSD) of 2.8% in 19 successive injections. The limits of detection of arginine (Arg), glucose, and methionine-glycine (Met-Gly) are estimated to be 2.0, 8.5, and 64.0 microM at S/N = 3, approximately 0.5-16.0 fmol, respectively. Variances influencing the separation efficiency and amperometric response, including injection, separation voltage, detection potential, or concentration of buffer and additive, are assessed and optimized.