To analyze umami peptides in the water extract of straw mushroom, fractionation guided by sensory evaluation and an electronic tongue of the extract was performed through sequential ultrafiltration and gel filtration chromatography. Fraction F1, which was found to make the greatest contribution to the overall umami, was further fractionated by reversed-phase high performance liquid chromatography combined with principal component analysis. Subfraction F1-a was found to have similar taste to straw mushroom. In addition the umami peptides from F1-a were identified by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The synthetic counterparts of the identified umami peptides were evaluated for their taste characteristics through sensory evaluation and electronic tongue analysis. The results showed that the umami peptides were identified as Asp-Asp-Cys-Pro-Asp-Lys, Leu-Val-Asp-Lys-Pro-Arg, Gln-Ala-Asp-Lys-Arg-Lys, and Asp-Thr-Phe-Asn-Asp-Lys, with umami taste threshold of 0.10, 0.33, 0.42, and 0.17 mg/mL, respectively. The flavor characteristics of these four umami peptides were attributed to the formation of specific structures by amino acids through peptide bonds. Among them, the umami peptide Asp-Asp-Cys-Pro-Asp-Lys was the most effective in enhancing the umami taste of monosodium glutamate (MSG) solution at a concentration of 20 mg/mL.