The comprehensive utilization and deep processing of freshwater fish resources are key research focuses. This study investigated the fermentation of Bighead carp meat with Mucor to enhance its umami quality and identified novel high-quality umami peptides. A total of 558 peptides were identified, and 113 potential umami peptides were screened through molecular docking with the taste receptor T1R1/T1R3. The top three peptides, based on molecular docking scores (NDLKDTSGKVVAK, AFLSDDKTKLELR, and VKKLVEAGAK), were chemically synthesized. Their umami taste was evaluated using an electronic tongue and sensory evaluation, with NDLKDTSGKVVAK exhibiting a significant umami flavor. Molecular dynamics simulations revealed that NDLKDTSGKVVAK shares a similar umami-inducing mechanism with other umami ligands. Hydrogen bonding and hydrophobic interactions were identified as the key interaction forces. Significant structural changes occurred in the protein around the cavity between the two subunits of T1R1/T1R3, involving key amino acid residues such as Glu736, Asp743, Arg747, and Glu744. • Umami quality of Bighead carp meat was enhanced after Mucor fermentation. • Umami peptide NDLKDTSGKVVAK was screened and characterized. • Umami forming mechanism of NDLKDTSGKVVAK was identical with other umami ligands.