In present work, 1,4-dioxane/Na2SO4 two-phase extraction system was developed for the separation of lactic acid from the synthetic solutions and the mixture directly derived from corn stover. The mass fraction of 1,4-dioxane, Na2SO4 amount, and pH of the system were found to be main factors influencing the extraction efficiency. Under the conditions (13.5 wt% of Na2SO4, 28.0 wt% of 1,4-dioxane, pH at 1.68) optimized by Response Surface Methodology (RSM), the highest extraction efficiency of lactic acid in simulation solution was up to similar to 91%. ATR-IR characterization of extraction system indicated that the formation of hydrogen bonding between 0 atom of 1,4-dioxane and H atom of 0-11 in lactic acid predominantly contributed to the excellent extraction efficiency. Furthermore, 1,4-dioxane/Na2SO4 two-phase system also showed excellent extraction efficiency (90%) for lactic acid from the mixture directly derived from the chemical catalytic conversion of corn stover. The extracted lactic acid could be further transformed to calcium lactate by salification, and the remaining 1,4-dioxane was recycled and directly used for the next run.