Three-phase single drop microextraction (SDME) was in-line coupled to chiral CE of weakly basic amine compounds including amphetamine. SDME was used for the matrix isolation and sample preconcentration in order to directly analyze urine samples with the minimal pretreatment of adding NaOH. A small drop of an acidic aqueous acceptor phase covered with a thin layer of octanol was formed at the tip of a capillary by simple manipulation of the liquid handling functions of a commercial CE instrument. While the saline matrix of the urine sample was blocked by the octanol layer, the basic analytes in a basic aqueous donor phase were concentrated into the acidic acceptor drop through the octanol layer by the driving force of the pH difference between the two aqueous phases. The enantiomers of the enriched amines were resolved by using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector for the subsequent CE separation. From 10 min SDME with the agitation of the donor phase by a small stirrer retrofit to the CE instrument, enrichment factors were about a 1000-fold, yielding the LOD of 0.5 ng/mL for amphetamine. This low LOD value as well as the convenience of in-line coupled SDME make the proposed scheme well suited for the demanding chiral analysis of amphetamine-type stimulants.