The diagnosis of amyotrophic lateral sclerosis (ALS) requires both upper and lower motor neuron signs. However, quite a few patients with ALS lack the upper motor neuron sign during the disease. This study sought to investigate whether metabolites, including glutamate (Glu), N-acetyl aspartate (NAA), and gamma aminobutyric acid (GABA), in the supplementary motor area (SMA) measured by magnetic resonance spectroscopy (MRS), could be a surrogate biomarker for ALS. Twenty-five patients with ALS and 12 controls underwent 3.0-T MR scanning, which measured Glu, NAA, and GABA. Finally, receiver operating characteristic (ROC) curves were created and the area under curve (AUC) was calculated to assess the diagnostic power. Logistic regression analysis revealed the usefulness of both Glu and NAA for the differentiation of ALS from controls (Glu, P = 0.009; NAA, P = 0.033). The ratio of Glu to NAA or GABA was significantly increased in patients with ALS (Glu/NAA, P = 0.027; Glu/GABA, P = 0.003). Both the AUCs were more than 0.7, with high specificity but low sensitivity. The present findings might indicate that both the Glu/NAA and the Glu/GABA ratios in the SMA could be potential biomarkers for the diagnosis of ALS.