Detection of native amino acids was accomplished using a capillary electrophoresis (CE)-amperometric detection system, in which a single carbon fiber cylinder (CFC) working electrode instead of a carbon fiber disc (CFD) electrode was mounted in the end part of a poly(dimethylsiloxane) (PDMS)/glass hybrid microchannel. Similar to that using CFD working electrode, here, the electrochemical reduction reaction at the working electrode is also coupled from the separation high voltage system, the coupling degree is related to the in-channel length of the CFC. This property simplifies the fabrication procedure of the working electrode and also provides a convenient and sensitive means for the determination of non-electroactive ions by amperometry. The present detection mode is successfully used to electrochemically detect non-electroactive arginine (Arg), threonine (Thr), glutamic acid (Glu) and electroactive cysteine (Cys). Furthermore, by simply changing the detection potential, we can easily distinguish peak mobilities of electroactive amino acids from that of non-electroactive amino acids.