Salivary D-alanine (D-Ala) and D-proline (D-Pro) are of concern for their potential in the noninvasive diagnosis of gastric cancer (GC). Most reports have succeeded in determining the total concentration of D-Ala and D-Pro. However, for personalized diagnosis and better elucidation of the underlying specific correlation of D-Ala (or D-Pro) with GC, it is desirable to determine the specific concentration of D-Ala or D-Pro. Herein, we propose an enantiomer-specific tandem assay of D-Ala based on the colorimetric reaction between 2,4-dinitrophenylhydrazine and pyruvic acid generated from the deamination of D-Ala catalyzed by D-amino acid oxidase, which is easily distinguished from L-form amino acids, D-Pro, and many other species. A linear concentration range is established from 20 to 400 μmol/L with a limit of detection of 1.01 μmol/L. Real saliva sample tests reveal that the levels of D-Ala in GC cases are remarkably higher than those in healthy individuals, which offers a simple and low-cost strategy for GC diagnosis. Simultaneously, the total concentrations of D-Ala and D-Pro in saliva are determined. Hence, the concentration of D-Pro and the proportion of D-Ala could be calculated, which further provides more molecule-and individual-specific information. This research may offer a convenient method for noninvasive diagnosis of GC and pave a new route to explore the potentials of rare D-form amino acids in disease diagnosis and treatment.