Objective To investigate the clinical diagnostic value of serum heat shock protein (HSP) 90A, stress induced phosphoprotein 1 (STIP1) and transgelin 2 (TAGLN-2) levels in adenomyosis. Methods 126 patients with adenomyosis diagnosed and treated in the Obstetrics and Gynecology Hospital Affiliated to Fudan University from January 2020 to June 2022 were selected as adenomyosis group. 65 women who underwent physical examination in the hospital during the same period were selected as healthy control group. The influence on adenomyosis formation was analyzed by unifactor and multivariate analysis, the relationship among the serum HSP90A, STIP1 and TAGLN-2 levels, the severity and dysmenorrhea of adenomyosis were also observed, and the diagnostic efficacy was analyzed in the diagnosis of adenomyosis. Results The levels of pregnancy time, birth time, serum HSP90A, STIP1 and TAGLN-2 in adenomyosis group were significantly higher than those in healthy control group (P＜0.01), but there were no significant differences in age, BMI, induced abortion history, delivery history, TG, TC, HDL-C and LDL-C between the two groups (P＞0.05) Multivariate analysis showed that pregnancy time, birth time, serum HSP90A, STIP1 and TAGLN-2 levels were independent risk factors for adenomyosis (P＜0.01) The serum levels of HSP90A, STIP1 and TAGLN-2 were increased with the grade of adenomyosis (P＜0.01), and the levels in the group with dysmenorrhea were significantly higher than those in the group without dysmenorrhea (P＜0.01) The sensitivity of serum HSP90A, STIP1 and TAGLN-2 levels in the diagnosis of adenomyosis was 60.3%, 77.0% and 77.0%, the specificity was 92.3%, 89.2% and 76.9%, and the AUC was 0.836, 0.884 and 0.829, respectively. The sensitivity and specificity of the combined detection were 92.9%, 92.3% and AUC was 0.966, which were significantly higher than that of the HSP90A (z=5.269, P＜0.001), STIP1 (z=3.867, P＜0.001) and TAGLN-2 (z=5.286, P＜0.001).Conclusion HSP90A, STIP1 and TAGLN-2 are involved in the pathogenesis of adenomyosis and are the indicators of the severity of adenomyosis. Combined detection is helpful to improve the diagnostic efficiency of adenomyosis