To study the umami peptides derived from porcine bone soup, ultrafiltration fractions with molecular weight less than 1 kDa were screened by sensory analysis which showed higher umami intensity. Four potential umami peptides were identified from the screened fractions by Nano-LC-Q-TOF-MS/MS, among which FSGLDGAK, FAGDDAPR and FSGLDGSK were proved to have dominant umami taste by sensory evaluation and electronic tongue. The threshold of the three peptides ranged from 0.1 mM to 0.89 mM. In addition, FSGLDGSK had the highest umami intensity and exhibited a significant umami-enhancing effect in a 0.35% monosodium glutamate solution. The results of molecular docking simulation showed that the key binding sites of taste receptor type 1 member 1 (His71, Asp108 and Glu301) and taste receptor type 1 member 3 (Glu48, Ser104 and His145) were crucial to the interaction with the umami peptides. Besides, electrostatic interaction and hydrogen bond mainly contributed to the mechanism of umami taste.