Taking Opuntia ficus-indica L . seed oil as the research object, its chemical constituents was analyzed by gas chromatography-mass spectrometry (GC-MS). The antioxidant activities in vitro of the Opuntia ficus-indica L. seed oil were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical, superoxide radical, total reductive capacity and total antioxidant capacity as indicators. Its irritancy was evaluated by hen’s egg test on the chorioallantoic membrane and repeated open application test. The results show that 16 compounds with a mass fraction greater than 0.1% are identified from the Opuntia ficus-indica L. seed oil accounting for 94.02% of the total mass. Among them, the mass fraction more than 1%: linoleic acid (C18∶2) 60.42%, hexadecanoic acid (C16∶0) 15.20%, stearic acid (C18∶0) 5.57%, γ-sitosterol 4.34%, linolenic acid (C18∶2) 1.89%, oleic acid (C18∶1) 1.48%, and cis-7-hexadecenoic acid (C16∶1) 1.13%. The Opuntia ficus-indica L . seed oil has strong antioxidant activity capacity, the half clearance rate (IC50) of DPPH free radical, hydroxyl free radical, superoxide anion, total reducing power and total antioxidant capacity are 5.11, 0.79, 3.19, 1.12 and 1.49 g/L, respectively. The hen’s egg test on the chorioallantoic membrane and repeated open application test show that Opuntia ficus-indica L. seed oil is non-irritating.