Abstract: Central Yunnan yellow beef is favored by consumers because of its high protein content, tender intermuscular fibers and delicious taste. However, the structure and umami mechanism of its umami peptides remain unclear. To study the umami peptides and umami mechanism of yellow beef in central Yunnan, ultrafiltration technology combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the peptides in the water extract of yellow cattle meat from central Yunnan. Based on virtual screening, the characteristic umami peptides of Dianzhong cattle meat were identified, and their theoretical taste characteristics were predicted. Molecular docking was used to reveal the binding mechanism between umami peptides and the receptors T1R1/T1R3. The sensory evaluation showed that the water extract of beef with a molecular weight <1 kDa had the best umami taste. A total of 2609 peptides were identified using LC-MS/MS, and 124 potential umami peptides were obtained by virtual prediction. Four characteristic umami peptides (LRDW, DVF, APKIPEGE, and GVWD) with high umami activity, safety, and non-toxicity were selected based on the analysis of umami-active fragments and their physical and chemical properties. The molecular weight ranged from 379.17 to 839.44 Da. Molecular docking showed that the four umami peptides mainly bind to the T1R3 receptor by activating the key amino acid residues His145, Glu148, Ala302, and His278, with hydrogen bonding being the key force involved, followed by electrostatic and hydrophobic interactions. This study provides a theoretical basis for improving the structure-activity relationship and efficient screening of umami peptides, and also provides a theoretical basis for the in-depth development of local beef umami peptides.