Determination of malic acid (MA) in commercial drinks is imperative to assure the quality of taste and safety of the product to human health. In this study, a column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was employed to determine the content of d- and l-MA in wines. The developed method successfully detected both MA enantiomers in several wines with a pre-column fluorescence derivatization procedure. Additionally, the present HPLC system provided sufficient data with a small sample quantity. Moreover, the d-MA peak was detected in the chromatogram of wine samples without acidulants. Thus, we believe that the proposed method would benefit the wine-brewing process and facilitate quality certification in the wine industry.