Objective To explore the mediating role of intolerance of uncertainty (IU) and moderating role of the negative emotion differentiation in the influence of perceived stress on anxiety among college students from a cognitive perspective. Methods A total of 271 participants were surveyed using the perceived stress scale, intolerance of uncertainty scale, depression anxiety and stress scale (Chinese version), and the test on negative emotional differentiation. SPSS 22.0 was used to perform descriptive statistics and correlation analyses and to test the moderated mediation model. Results Perceived stress affected anxiety and IU played a mediating role－perceived stress could affect anxiety through influencing IU. At the same time, the influence of IU on anxiety could be adjusted through the negative emotion differentiation. The higher the degree of negative emotion differentiation, the lower the degree of anxiety increase (β＝0.17, t＝5.70, P＜0.01). Conclusion It may be effective to develop training programs to reduce anxiety by regulating perceived stress, increasing acceptance of uncertainty, and improving the negative emotion differentiation, which can help individuals reduce anxiety by perceiving and adjusting anxiety-related emotional or cognitive factors in a timely manner.