The aim of this study was to investigate the expression of macrophage migration inhibitory factor (MMIF), hypoxia-inducible factor-1 a (HIF-1α) and vascular endothelial growth factor (VEGF) in the serum and endometrial tissues of patients with endometriosis (EM) and the clinical significance. Eighty EM patients [American Reproductive Association stage I (n=20), stage II (n=22), stage III (n=21) and stage IV (n=17)] were enrolled and divided into mild (10-14 points, n=28), moderate (16-24 points, n=27) and severe (26-30 points, n=25) dysmenorrhea groups. The control group included 40 healthy women of childbearing age who underwent routine healthcare examinations in the enrolment period. The expression of MMIF, HIF-1α and VEGF in the serum and endometrial tissues was measured by enzyme-linked immunosorbent assay and Western blotting, respectively. Meanwhile, the sensitivity and specificity of serum MMIF, HIF-1α, and VEGF when separately used as single indexes or jointly used as one index were examined as well. The results showed that serum concentrations of MMIF, HIF-1α, and VEGF were significantly higher in EM patients than in controls (P<0.05). The expression of all three proteins in both serum and endometrial tissues increased significantly with the R-AFS stage (P<0.05) and with dysmenorrheal severity (P<0.05). The sensitivity and specificity of the combined detection of serum MMIF, HIF-1α, and VEGF levels were significantly higher than those of single index detection (P<0.05). In conclusion, the expression of MMIF, HIF-1α, and VEGF in the serum and endometrial tissues may be used to assess the stage of EM and the severity of dysmenorrhea. Combined evaluation of MMIF, HIF-1α, and VEGF significantly improves the diagnostic sensitivity and specificity.