A mixture of nine biphenyl nitrile compounds with high hydrophobicity and similar structures was successfully separated by microemulsion electrokinetic chromatography (MEEKC) within 30 min. The buffer system contained 100 mmol/L sodium dodecyl sulfate (SDS), 80 mmol/L sodium cholate (SC), 0.81% heptane, 7.5% n-butanol, 10% acetonitrile and 10 mmol/L borate. The addition of SC, organic modifiers, sample preparation and temperature all showed remarkable effect on the separation. Meanwhile, the MEEKC method was briefly compared with micellar electrokinetic chromatography (MEKC) method. A mixture of nine biphenyl nitrile compounds with high hydrophobicity and similar structures was successfully separated by microemulsion electrokinetic chromatography (MEEKC) within 30 min. The buffer system contained 100 mmol / L sodium dodecyl sulfate (SDS), 80 mmol / L sodium cholate ), 0.81% heptane, 7.5% n-butanol, 10% acetonitrile and 10 mmol / L borate. The addition of SC, organic modifiers, sample preparation and temperature all showed remarkable effect on the separation. Meanwhile, the MEEKC method is briefly compared to with micellar electrokinetic chromatography (MEKC) method.