Hydroxy acids are compounds referred to as organic acids with a hydroxyl group, and are widely used in industries such as medicine, food, cosmetics, and chemical products. 3-Hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis can catalyze 3-oxobutyrate, also known as acetoacetate. Therefore, using rational design with mutational studies, 3HBDH had been engineered to convert 4-oxovalerate, also known as levulinic acid, into 4-hydroxyvalerate. In this study, activities for keto acid substrates with variable carbon chain length (C3-C8) were measured, and substrate specificity was analyzed through structural analysis. The engineered 3HBDH was able to react with all candidate substrates, while the wildtype 3HBDH reacted with only a few. Analyzing the distance between the substrate and the enzyme by docking simulation, Ser142 and Tyr155 were directly related to the catalytic mechanism. In addition, Gln196 appeared to be involved in the enzyme-substrate complex by binding hydrogen bonds with ketone and carboxyl groups of the substrate. Based on the results of this study, data for engineering strategy of a redox enzyme platform for synthesizing organic acids were obtained.