Abstract: This study aims to provide a theoretical foundation and technical support for the development of Angiotensin-converting enzyme（ACE）inhibitory peptides derived from Hongqu rice distiller’s grains. ACE inhibitory peptides were obtained from Hongqu rice distiller’s grains, its functional structure and stability in vitro digestion were studied. The preparation process of ACE inhibitory peptide was optimized by single factor and orthogonal test. The enzymatic hydrolysate was separated and purified by ultrafiltration. The functional structure of the active peptide was determined by mass spectrometry, and the stability of in vitro digestion was analyzed by simulated gastrointestinal digestion. The optimum preparation process of ACE inhibitory peptides was as follows: enzyme addition amount 6000 U·g⁻¹, 90 min, 45℃, pH 5.7. Under these conditions, the enzymatic hydrolysis rate was（45.11 ± 0.12）%, and the ACE inhibition rate was（60.49 ± 1.70）%, which was mainly composed of short peptides with molecular weight less than 3000 Da. The enzymatic hydrolysis products were subjected by ultrafiltration, and components with less than 1 000 Da showed higher ACE inhibitory activity, with an inhibition rate of（70.65 ± 1.51）%. The proportion of potential bioactive peptides with a biological activity value ≥0.5 is 48.22%. After simulating gastrointestinal digestion, the activity retention rate was 58.94%. The preparation of ACE inhibitory peptides from Hongqu rice distiller’s grains by papain provides a basis for the development of antihypertensive functional food ingredients and the high-value utilization of wine by-products.