Background <p>Dysmenorrhea is a common gynecological condition with heterogeneous etiologies. Endometriosis is an important condition associated with dysmenorrhea, but the serum steroid and reproductive hormone profiles related to dysmenorrhea, and whether these associations differ by endometriosis status, remain unclear. This study aimed to investigate the associations of serum hormone profiles with dysmenorrhea and pain severity.</p> Methods <p>This exploratory cross-sectional study was conducted from October 2022 to January 2024 at Zhongshan Hospital, Fudan University. A total of 139 premenopausal participants were classified into dysmenorrhea (<i>n</i> = 73) and non-dysmenorrhea (<i>n</i> = 66) groups and further stratified by endometriosis status. Serum steroid and reproductive hormones were measured using liquid chromatography–tandem mass spectrometry and immunoassays. Group comparisons were performed according to data distribution, and false discovery rate (FDR) adjustment was applied for multiple hormone comparisons. Multivariable logistic regression was used to evaluate the association between androstenedione (A4) and dysmenorrhea.</p> Results <p>In the overall cohort, women with dysmenorrhea showed higher A4 levels and a higher luteinizing hormone/follicle-stimulating hormone (LH/FSH) ratio, although these differences did not remain significant after FDR adjustment. After stratification by endometriosis status, these differences were mainly observed among women without endometriosis. In this subgroup, A4 levels were significantly higher in women with dysmenorrhea than in those without dysmenorrhea (0.85 ± 0.36 vs. 0.65 ± 0.28 ng/mL, <i>P</i> = 0.004; FDR-adjusted <i>P</i> = 0.024), as was the LH/FSH ratio (2.11 ± 1.47 vs. 1.44 ± 1.02, <i>P</i> = 0.013; FDR-adjusted <i>P</i> = 0.047). Among women without endometriosis, A4 was modestly correlated with visual analogue scale score (Spearman r = 0.285, <i>P</i> = 0.006) and remained associated with dysmenorrhea after multivariable adjustment (odds ratio = 8.07, 95% confidence interval: 1.60–40.65, <i>P</i> = 0.011). A4 and the LH/FSH ratio showed modest discrimination, with area under the curve values of 0.660 and 0.655, respectively.</p> Conclusions <p>Serum A4 levels were associated with dysmenorrhea and pain severity, particularly among women without endometriosis. The LH/FSH ratio showed a weaker supportive association. These exploratory findings support further investigation of androgen-related endocrine profiles in dysmenorrhea without endometriosis, but A4 and the LH/FSH ratio showed limited standalone discriminatory value.</p> Trial registration <p>Chinese Clinical Trial Registry, ChiCTR2400079587, Retrospectively registered on 2024–01-08.</p>