This study compares umami peptides prepared by trypsin hydrolysis and boiling and analyzes their umami intensity and characteristics. Using a taste reconstitution model and taste evaluation analysis, the study revealed that umami peptides prepared by boiling have a higher umami contribution. Myosin and heat shock protein were identified as marker proteins for revealing differences of cleavage sites. Boiling releases a higher proportion of acidic amino acids at the protein cleavage sites p1-p1', whereas trypsin hydrolysis releases more basic amino acids. Molecular docking simulation and electrostatic potential observation showed that acidic amino acid residues have a wider binding range with the umami receptor T1R1-VFT. Acidic amino acids lower the isoelectric point (pI) of umami peptides, enhancing their negative charge at pH 7, which are more likely to bind to the positively charged regions of the umami receptor.