In this study， the enzymatic hydrolysis preparation of flavor peptide from Stropharia rugosoannulata were optimized by using response surface methodology based on the taste characteristics and peptide yield. The optimal process parameters were as follows， the hydrolysis time was 51.62 min， the addition amount of alkaline protease was 0.99% (enzyme activity， 200 000 U/g)， the hydrolysis temperature was 42.03 ℃， and the substrate mass concentration was 48.45 g/L. Based on the optimal parameters of conventional enzymatic hydrolysis process， the peptide was prepared by simultaneous ultrasound-assisted enzymatic hydrolysis. The peptide content in the samples(469.21 mg/g dry weight) at 30 min treatment of the plate ultrasound-assisted enzymatic hydrolysis could reach the peptide yield under the optimal conditions of conventional enzymatic hydrolysis， and the preparation time of the base material was significantly reduced； the peptide yield (492.87 mg/g dry weight) at 40 min treatment of ultrasound-assisted enzyme hydrolysis was 6.5% higher than that of conventional enzyme hydrolysis. The peptides prepared by simultaneous ultrasound-assisted enzymatic hydrolysis had better savory and umami taste characteristics， which were enhanced by 1.1-1.8 times and 1.1-1.3 times， respectively， compared with the peptide prepared by conventional enzymatic hydrolysis. The peptide-rich samples showed superior angiotensin-I converting enzyme (ACE) inhibitory activity with 50% inhibitory concentration (IC50) of 0.071， 0.074， 0.122 mg/mL and 0.123 mg/mL. By analyzing the sequence of the flavor peptide， it was found that the peptide showed salty and umami taste when the aspartic acid (D)， valine acid (V)， glutamic acid (E)， leucine acid (L)， glycine acid (G)， arginine acid (R)， proline acid (P)， and alanine acid (A) in the combination of DD， VV， EL， VG， VE， RP， RA， RV or VR were present in the peptide sequence. The high proportion of these amino acids' combination in the peptides might be the main reason for the salty and umami taste characteristics of the samples. The mechanism of in vitro ACE inhibitory activity of the flavor peptide was predicted by molecular docking technique， and the flavor peptides could form hydrogen bond interaction with the amino acid residues of ACE active pocket， which might be the main reason for the ACE inhibitory effect of the flavor peptides. The study can provide a theoretical basis for the preparation and application of the flavor-active peptide of mushroom， and provide a reference for the preparation of salt-reducing and umami-enhancing condiments， functional foods， or food additives.