Abstract: An online focusing method, based on fieldenhanced sample injection coupled with transient moving substitution boundary, was developed for the separation and capillary electrophoresis(CE)amperometric detection (AD) of biogenic amines under acidic conditions for the first time. The proposed method was based on the substitution reaction between 18crown6bonded biogenic amine complexes and Na+ cations at the substitution boundary. By using this method, electrode fouling was prevented in electrochemical detection. The factors affecting extraction efficiency, separation, and detection were investigated. Under the optimum conditions, the limits of detection of five analytes in a sample, which was prepared in a lowconductivity matrix, ranging from 2.2 to 9.8 nmol/L (signaltonoise ratio, S/N＝3), and about 160 to 300fold enhancement in concentration was achieved compared to the conventional CEAD method. This method has great potential for the detection of biogenic amines.