The DNA-based selector for discriminating chiral ofloxacin with high enantioselectivity and affinity is constructed through Cu(II)-coordination with G-rich duplex containing successive guanines. Using this chiral selector, R- and S-ofloxacin can be directly enriched from the racemate, with the enantiomeric excess of 85% (R) and 78% (S) individually by three operational stages.