The influences of the composition of microemulsion on the microstructure including dimensions and zeta potentials of microdroplets were measured in details. The average dynamic dimension of microdroplets was measured by dynamic laser light scattering, and zeta potential was determined to characterize average surface charge density of microdroplets. The experiment results showed that increase of the amount of surfactant resulted in decrease of microdroplet size but almost invariant zeta potential, which would enlarge migration time of the microdroplet in MEEKC. With increment of cosurfactant concentration, the microdroplet size had an increasing trend, whereas the zeta potential decreased. Thus, observed migration velocity of microdroplets increased, which made the separation window in MEEKC shortened. Neither dimension nor zeta potential of microdroplets changed by varying both the type and the amount of the oil phase. Adding organic solvent as modifier to microemulsion did not change the microdroplet size, but lowered zeta potential. The migration time of microdroplet still became larger, since EOF slowed down owing to organic solvent in capillary. So, besides increment of surfactant concentration, organic additive could also enlarge the separation window. Increase of cosurfactant concentration was beneficial for separation efficiency thanks to the looser structure of swollen microdroplet, and the peak sharpening might compensate for the resolution and peak capacity owing to a narrow separation window. Except the oil phase, tuning the composition of microemulsion would change the microstructure, eventually could be exploited to optimize the resolution and save analysis time in MEEKC.