Objective: This study focused on the fatty acid composition and antioxidant activity of watermelon seed oil. Methods: The watermelon seed oil was extracted by Soxhlet extraction from the watermelon seed, then the watermelon seed oil was esterified. Thereafter, the components and the contents of watermelon seed oil were analyzed and detected used the method of gas chromatography-mass spectrometry (GC-MS), and the antioxidant activity of watermelon seed oil was studied by measuring its ability to reduce iron ions and scavenge DPPH radical, hydroxyl radical and superoxide anion. Results: The results of GC-MS showed that there were 6 fatty acids in watermelon seed oil, the relative content of unsaturated fatty acidswas more than 50%, the study of antioxidant activity showed that watermelon seed oil had the ability to reduce iron ions, and the IC₅₀ value of watermelon seed oil for eliminating DPPH free radical, superoxide anion and hydroxyl free radicals was 3.7 mg/mL, 0.031 mg/mL and 12.04 mg/mL, respectively. Conclusion: Watermelon seed oil is rich in unsaturated fatty acids and has good antioxidant activity.