Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. However, currently available methods are time consuming, potentially delaying diagnosis. Therefore, the development of more rapid analytical tools is needed. In this study, a chemiluminescence detection method for amino acids was developed, and the conditions for the enzyme reaction and assay were examined. For the recognition of each amino acid (here, serine, proline, glycine, asparagine, leucine, and histidine), the corresponding aminoacyl-tRNA synthetase (aaRS) was employed, and multiple enzymatic reactions were combined with a luminol chemiluminescence reaction. This method provided selective quantification from 1 to 20 mu M for serine, proline, glycine, and leucine; 1 to 60 mu M for asparagine; and 1 to 150 mu M for histidine. This assay, which utilized aaRSs for the detection of amino acids, could be useful for simple and rapid analysis of amino acids in clinical diagnostics.