Semen Allii Fistulosi (PSAF) is the seed of Allium fistulosum L. of the Liliaceae family. The purpose of this study was to extract, characterize, and evaluate the antioxidant activity in vitro of proteins. Using single factor and orthogonal design, the optimum conditions of extraction were determined to be as follows: extraction time 150 min, pH 8.5, temperature 60 °C, and ratio (v/w, mL/g) of extraction solvent to raw material 35. The isoelectric point of the pH was determined to be about 4.4 and 10.2, by measuring the protein content of PSAF solutions at different pH values. The amino acid composition of PSAF was determined by high performance liquid chromatography (HPLC), and the results suggested that the species of amino acids contained in the PSAF was complete. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed the molecular weight was mainly between 40 and 55 kDa, and Fourier-transform infrared spectroscopy (FTIR) characterized prevalent protein absorption peaks. PSAF exhibited potent scavenging activities against DPPH assays, via targeting of hydroxyl and superoxide radicals, while chelating Fe2+ activity and demonstrating weak reducing power. This work revealed that PSAF possessed potential antioxidant activity in vitro, suggesting potential for use of PSAF as a natural antioxidant.