Objective This study analyzed the expression of alpha-hydroxybutyrate dehydrogenase (α-HBDH) in serum and explored its predicative value in the diagnosis of ovarian cancer (OC). Methods A retrospective study was conducted on 319 OC patients (OC group), 400 patients with benign lesions (benign group), and 400 healthy controls (normal group). These subjects were treated or received physical examination in Women's Hospital, School of Medicine, Zhejiang University from January 2014 to August 2018. Each group was further stratified by menopausal status. The expression levels ofα-HBDH and carbohydrate antigen125 (CA125) and their associations with clinic-pathological characteristics of OC were evaluated, and their diagnostic efficacy in OC were explored. Wilcoxon Rank Test and Kruskal-Wallis H test were used for group comparison. Receiver operating curve(ROC) was plotted to evaluate the diagnostic capability of α-HBDH and CA125 in OC. Results The median of α-HBDH level was 134.0 U/L in OC group, 120.0 U/L in benign group, and 110.0 U/L in normal group. OC group was significantly different from other two groups (H=129.5, P<0.001). Serumα-HBDH was also correlated with the menopausal status, lymph node metastasis, and clinical stage of OC patients significantly(Z=-5.2, H=31.5,Z=-3.2,all P<0.001). In the ROC analysis in terms of OC risk, the area under curve (AUC) ofα-HBDH was lower than AUC of CA125 [premenopausal group (α-HBDH: AUC=0.685; CA125: AUC=0.796;menomenopausal group (α-HBDH:AUC=0.749;CA125:AUC=0.915)];and in the stage I of premenopausal group, α-HBDH performed similar to CA125, but with obviously higher sensitivity than CA125 (α-HBDH:AUC:=0.646, Se=79.41％, SP=41.61％;CA125:AUC=0.691, Se=58.82％, Sp=74.71％). Conclusions The expression level of serum α-HBDH level was increased in OC patients, and it was associated with menopausal stage, lymph node metastasis, and clinical stage of OC. In addition, α-HBDH showed higher sensitivity than CA125 in stage I premenopausal group, which was potentially beneficial for the diagnosis of stage I OC in premenopausal women.