A simple, sensitive and rapid spectrofluorimetric method for determination of α-tocopherol in pharmaceutical capsule and human plasma was developed and validated. The native fluorescence of α-tocopherol was measured at 334 nm with excitation at 291 nm, after extraction of α-tocopherol from human plasma hexane:dichloromethane mixture. The calibration curves were linear (R≥0.9993) in the concentration range of 0.25-2.5 μg/ml of α-tocopherol in both standard solutions and plasma samples. The developed method was directly and easily applied for determination of α-tocopherol in the plasma of healthy volunteers and different type of bladder cancer and stomach cancer patients and also pharmaceutical capsule.