Objective To explore the mediating effect of dyadic coping between perceived stress and negative emotions in spouses of female patients with infertility undergoing in vitro fertilization⁃embryo transfer. Methods A total of 208 spouses of female patients with infertility who received in vitro fertilization⁃embryo transfer were selected as the research subjects. Investigations were performed on them by using the Perceived Stress Scale (PSS), Dyadic Coping Inventory (DCI), and Hospital Anxiety and Depression Scale (HADS). The correlation between total scores of the 3 scales was analyzed. The Model 4 of PROCESS macro for SPSS software was employed to analyze the mediating effect, and the mediating effect was validated by using the Bootstrap method. Results The total scores of PSS, DCI, and HADS among 208 spouses of female patients with infertility were 14.26±4.89, 123.95±16.34, and 11.43±5.01, respectively, therein the incidence rates of anxiety and depression were 36.06% (75/208) and 24.52% (51/208), respectively. The results of correlation analysis revealed that PSS total score positively correlated with HADS total score in spouses of female patients with infertility, whereas negatively correlated with DCI total score; in addition, DCI total score negatively correlated with HADS total score (P<0.05). The results of mediating effect analysis and validation indicated that dyadic coping exerted a partial mediating effect between perceived stress and negative emotions, and the mediating effect value was 0.058 (0.013, 0.112), accounting for 9.7% of the total effect. Conclusion Perceived stress of spouses of female patients with infertility is at the medium level. Perceived stress can directly and positively affect negative emotions, or it can indirectly affect negative emotions through dyadic coping. Therefore, medical staff can improve dyadic coping ability of spouses of female patients with infertility to reduce the occurrence of their negative emotions.