Natural standard grade γ-tocopherol are of great interest due to its good antioxidant activity and special pharmacological functions. This study separated and purified γ-tocopherol from mixed tocopherols using eco-friendly and low toxicity solvents. 0.28 g γ-tocopherol was obtained per performance with a purity of 98.89 ± 0.68% and a recovery yield of 93.23 ± 0.89%. The purification conditions were as follows: elution solvent n-hexane/ethyl acetate 94.5:5.5 (v/v), sample load size 0.5 g, a column height to diameter ratio of 16:1 and an elution rate of 2 mL/min. The purity and structure of γ-tocopherol was confirmed by HPLC, GC/MS and NMR. The adsorption isotherm of γ-tocopherol on silica gel (200-300 mesh) fitted the Langmuir isotherm model well. The antioxidant activity analysis showed that γ-tocopherol had the strongest antioxidant activity followed by δ-tocopherol and α-tocopherol. This study provides an economically attractive solution for the production of natural standard grade γ-tocopherol.