Abstract: The anti-oxidation of natural extracts of rosemary, tea polyphenols, vitamin E and TBHQ in the cotton seed oil was studied by Rancimat method. With adding different mass concentrations of single antioxidant and different ratio of antioxidant combination into cotton seed oil, the anti-oxidation effects were determined by testing their induction time. The results showed that the anti-oxidation of natural antioxidants in cotton seed oil listed descending as TBHQ>rosemary>tea polyphenols>vitamin E. In the complex combination of antioxidants, rosemary extract and tea polyphenols by 3:1 combination showed good synergistic effect. The antioxidant effect was better than the same mass concentration of natural antioxidants when the natural antioxidants combined with TBHQ.