Abstract: Vacuum extraction method was selected to improve the extraction efficiency of total saponins from Sapindus mukorossi Gaertn with water as extraction solvent. The contents of total saponins were determined by spectrophotometry and ultra-performance liquid chromatography(UPLC). Based on single factor experiments, orthogonal design was used to optimize the process parameters. The optimized extraction process was extraction temperature 60 ℃, extraction time 1 h and the solid-to-liquid ratio 1:14(g:mL). Under these conditions, the extraction rate of total saponins in S. mukorossi fruits was 15.34% by using spectrophotometry. The herbicidal activity of the extract was evaluated by water agar medium method. The fruit extract exhibited obvious root-growth inhibition activity against Trifolium pratense L. with EC₅₀ value of 0.116 g/L. The herbicidal active compounds were tentative identified as triterpenoid saponins by thin-layer chromatography(TLC).