A new phenylalanine derivative (l-N-(2-hydroxy-propyl)-phenylalanine, l-HP-Phe) was synthesized and its chelate with Cu(II) (Cu(II)–(l-HP-Phe)2) was used as the chiral selector for the ligand-exchange (LE) chiral separation of d,l-selenomethionine (SeMet) in selenized yeast samples by micelle electrokinetic capillary chromatography (MEKC). In order to improve the sensitivity of MEKC–UV, two-step preconcentration strategy was employed, off-line solid phase extraction (SPE) and on-line large volume sample stacking (LVSS). d,l-SeMet was first retained on the Cu(II) loaded mesoporous TiO2, then eluted by 0.1mL of 5molL−1 ammonia, and finally introduced for MEKC–UV analysis by LVSS injection after evaporation of NH3. With the enrichment factors of 1400 and 1378, the LODs of 0.44 and 0.60ngmL−1 for l-SeMet and d-SeMet was obtained, respectively. The developed method was applied to the analysis of d,l-SeMet in a certified reference material of SELM-1 and a commercial nutrition yeast, and the results showed that most of SeMet in the SELM-1 selenized yeast was l isomer and the recovery for l and d isomers in the spiked commercial nutrition yeast was 96.3% and 103%, respectively. This method is featured with low running cost, high sensitivity and selectivity, and exhibits application potential in chiral analysis of seleno amino acids in real world samples.