Wuding chicken is famous for its delicious meat, and HLEEEIK, LDDALR, and ELY were jointly extracted from different processing stages of Wuding chicken. However, whether these peptides can be used as umami supplements is unclear. The sensory evaluation tests were used to study the taste characteristics. The secondary structure of the peptides and their interaction with T1R1/T1R3 were predicted by the circular dichroism spectrum and molecular dynamics simulation. The umami threshold was 0.03125 to 0.06250 mg/mL, all of which could increase umami, saltiness, sweetness, and mask bitterness. Compared with HLEEEIK, the frequency of umami active fragments and the improvement rate of the umami score of EEE increased by 133.35% and 40.09%, respectively. Peptides were dominated by umami taste according to sensory analysis, among which EE-3 (3.18) has the highest umami intensity followed by LR-4 (2.58), HK-7 (2.13), and EY-3 (1.82). The main secondary structure of umami peptides was β-folding, and Tyr74, Arg323, Arg272, and Gln35 were the key amino acid residues for binding of umami peptides to the receptor. This study further elucidated that the umami intensity of the peptides could be altered by changing the sequence composition of the peptides, which enhanced our understanding of the complex flavor properties of umami peptides.