Distinguishing between the two enantiomers of a molecule is a challenging task due to their nearly identical physical properties. Time-consuming chromatography methods are typically required for this task, which greatly limits the throughput of analysis. Here we describe a fluorescence-based method for the rapid and high-throughput analysis of both small-molecule enantiopurity and concentration. Our approach relies on selective molecular recognition of one enantiomer of the target molecule using a DNA aptamer, and the ability of aptamer-based biosensors to transduce the presence of a target molecule into a dose-dependent fluorescence signal. The key novel aspect of our approach is the implementation of enantiomeric DNA biosensors, which are synthesized from D- and L-DNA, but labeled with orthogonal fluorophores. According to the principle of reciprocal chiral substrate specificity, these biosensors will bind to opposite enantiomers of the target with equal affinity and selectivity, enabling simultaneous quantification of both enantiomers of the target. Using the previously reported DNA biosensor for L-tyrosinamide (L-Tym), we demonstrate the ability to rapidly and accurately measure both enantiopurity and concentration for mixtures of l- and D-Tym. We also apply our enantiomeric biosensors to the optimization of reaction conditions for the synthesis of D-Tym and provide mathematical modeling to suggest that DNA biosensors having only modest binding selectivity can also be used for fluorescence-based enantiopurity measurement. This research provides a generalizable method for high-throughput analysis of reaction mixtures, which is anticipated to significantly accelerate reaction optimization for the synthesis of high-value chiral small molecules.