Human aldo-keto reductase AKR1C3 (type 2 3α-hydroxysteroid dehydrogenase/type 5 17β-hydroxysteroid dehydrogenase) is involved in testosterone and estrogen metabolism. AKR1C3 expression is relatively low in most tissues and high in prostate and mammary glands in regulating androgen and estrogen levels. However, in many cancers, overexpression of AKR1C3 was observed, thus prompting the development of therapeutics targeting AKR1C3. To facilitate the development of AKR1C3 targeting therapeutics, evaluating the expression of AKR1C3 is vital. As AKR1C3 is highly homologous with its family proteins, AKR1C1, AKR1C2, AKR1C4 and other AKR1 proteins, reagents that can unambiguously discriminate these enzymes are needed. In this report, a highly specific monoclonal antibody for AKR1C3, 10B10, was developed and characterized. Compared to other AKR1C3 antibodies, 10B10 is highly specific and sensitive to AKR1C3 in multiple assay formats. Thus, 10B10 will be a valuable tool for the clinical development of AKR1C3 targeting therapeutics and the study of AKR1C3 biology.