Abstract: OBJECTIVE To optimize the extraction process of Menispermi Rhizoma fatty oil by Box-Behnken response surface methodology and analyze its composition and study the antioxidant activity of Menispermi Rhizoma fatty oil in vitro. METHODS The extraction rate of Menispermi Rhizoma fatty oil as an index and based on the single-factor tests, Box-Behnken response surface methodology was used to investigate the effects of extraction temperature, extraction time and material-liquid ratio on extraction process; the composition of fatty oil was analyzed by GC-MS; the 1,1-diphenyl-2-picrylhydrazyl(DPPH) free radical experiment was used to evaluate the in vitro antioxidant activity of Menispermi Rhizoma fatty oil. RESULTS After optimization with the response surface methodology, it was determined that the optimal extraction process was extraction temperature of 90℃ and reflux extraction of 3 times for 2.3 h each time, the ratio of solid to liquid was 1:21. Under these conditions, the extraction rate was 1.820%; there were 32 components identified from Menispermi Rhizoma fatty oil by GC-MS. The total antioxidant activity of Menispermi Rhizoma fatty oil increased gradually with the increase of the concentration, after the concentration reached 0.833 mg·mL^-1, the DPPH free radical scavenging rate increased slowly, at the concentration of 1.333 mg·mL^-1, the scavenging rate was 70.1%. CONCLUSION The optimized process is convenient, stable and feasible, which can provide references for subsequent research. Menispermi Rhizoma fatty oil contains many important fatty oil components and has certain antioxidation activity in vitro.