A capillary modified by assembling a molecular film was presented for the chiral separation of sertraline by microemulsion electrokinetic chromatography. The assembling molecular film was constructed with poly(diallyldimethylammonium-chloride) and β-cyclodextrin via inclusion complexation. The separation efficiency of cis-trans isomers and enantiomers of sertraline was improved with a running microemulsion that contained the acetonitrile, sodium dodecyl sulfate, n-butanol and n-hexane buffered with sodium tetraborate. The baseline separation of four sertraline cis-trans isomers and enantiomers was achieved under the optimum conditions. The detection limit for isomers and enantiomers of sertraline (1S,4S, 1R,4R, 1S,4R, 1R,4S) was 0.15, 0.15, 0.30, 0.30 mg/L, respectively. The mechanism of chiral separation was studied and it could be applied for the determination of commercial Zoloft tablet samples satisfactorily.