〔Abstract〕 Objective To investigate the expression patterns of key steroidogenic enzymes (CYP11A1, HSD3B1, and CYP19A1) in placental tissues from preeclampsia (PE) patients and evaluate their potential as biomarkers for PE. Methods A case-control study was conducted using placental tissues from PE patients (PE group, n=3) and normotensive pregnant women (control group, n=3). RNA sequencing was performed to identify differentially expressed genes (DEGs), followed by validation of target gene expression using Western blot, qRT-PCR, and immunohistochemistry. Steroid hormone levels in placental tissues and serum were measured by ELISA. Spearman correlation analysis was used to assess associations between gene expression and clinical parameters, and receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive value of target genes for PE. Results The results showed that 501 DEGs (P<0.05 and |Log2fold change| >1) were identified in PE placental tissues, with KEGG enrichment analysis revealing significant involvement in steroid hormone biosynthesis pathways. Compared with the control group, PE placental tissues exhibited significantly decreased protein and mRNA expression of HSD3B1 and CYP19A1 (P< 0.01), while CYP11A1 expression was upregulated (P<0.05). Serum estradiol (E2) and progesterone (P4) levels were significantly reduced in the PE group (P<0.01), whereas testosterone (T) levels were elevated (P<0.05). Correlation analysis demonstrated that CYP11A1 expression was positively associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), and soluble fms-like tyrosine kinase-1 (sFlt-1) (P<0.01), but negatively correlated with placental growth factor (PLGF). Conversely, HSD3B1 and CYP19A1 expression showed inverse correlations with these clinical parameters. ROC analysis revealed that CYP11A1, HSD3B1, and CYP19A1 each had an area under the curve (AUC) of 0.778 for predicting PE, with 95%CI ranging from 0.614 to 0.942 (P=0.004). The sensitivity was 94.4%, and the specificity was 66.7%. Conclusion These findings suggest that dysregulated expression of steroidogenic enzymes in PE placental tissues contributes to hormonal imbalance, thereby playing a role in PE pathogenesis. Furthermore, CYP11A1, HSD3B1, and CYP19A1 may serve as potential diagnostic biomarkers for PE.