The objective of this study was to investigate the umami characteristics of soy sauce using electronic tongue evaluation and amino acid composition and Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The soy sauce peptides were isolated from soy sauce using XAD-16 macroporous resin combined with ethanol solution. Results showed that the soy sauce peptide fraction eluted by 60% ethanol (SS-60%) exhibited a prominent umami taste and the umami scores were highly positively correlated with the amino acid nitrogen contents of soy sauces. The umami scores of SS-60% were significantly positively correlated with the contents of free amino acids. Especially, Phe showed the highest positive correlation with the umami scores. In addition, five characteristic ion peaks with m/z at 499, 561, 643, 649, and 855 were identified in the peptide mass fingerprinting. Therefore, this study provides new insights into the umami characteristics for the flavor evaluation and reality identification of soy sauce.