An on-line technique for pressure and electrokinetic injections of long sample plugs with simultaneous stacking of neutral analytes (notoginsenoside R-1, ginsenoside Rg(1), ginsenoside Rf, ginsenoside Rh-1, ginsenoside Rd, ginsenoside Rg(3)) in microemulsion electrokinetic chromatography is presented. The effects of salt concentration, sample plug length, organic modification of the sample matrix, oil phase and SDS concentration on stacking efficiency were examined in order to optimize the two injection methods. in microemulsion electrokinetic chromatography, the effect of the type of oil and SDS content on stacking mechanism is often sophisticated. This study had demonstrated that the oil type and SDS content in microemulsion indeed markedly altered the affinity of microemulsion with analytes. Finally, in comparison with the electrokinetic injection method, the most apparent disadvantages of the pressure injection method were the relatively high LOD and poor reproducibility.