Abstract: In order to provide theoretical basis and technical support for the development of Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from the fermented red rice distiller's grains, the ACE inhibitory peptide was prepared from the fermented red rice distiller's grains, and the bioactive peptides sequence and digestion stability were determined. The preparation process of ACE inhibitory peptides was optimized by single factor and orthogonal test. The ACE inhibitory peptides were isolated and purified by ultrafiltration. The sequential structure of the active peptides was analyzed by mass spectrometry, and the stability of in vitro digestion was analyzed by simulated gastrointestinal digestion. The results showed that: the optimum preparation process of ACE inhibitory peptides from the fermented red rice distiller's grains was as follows: enzyme addition amount of 6 000 U·g⁻¹, enzymatic time of 90 min, temperature of 45℃, and pH of 5.7. Under these conditions, the enzymatic hydrolysis rate of the fermented red rice distiller's grains protein was (45.11 ± 0.12)%. When the protein concentration was 2.7 mg·mL⁻¹, the ACE inhibition rate was (60.49 ± 1.70)%, which was mainly composed of short peptides with the molecular weight less than 3000 Da. The enzymatic hydrolysates were subjected by ultrafiltration, and the components with less than 1 000 Da had higher ACE inhibitory activity, with an inhibition rate of (70.65 ± 1.51)%. The potential bioactive peptides with the biological activity value ≥ 0.5 accounted for 48.22%. After the simulated gastrointestinal digestion, the ACE inhibitory activity retention rate of the components with ACE inhibitory peptides less than 1 000 Da was 58.94%. The preparation of ACE inhibitory peptides from the fermented red rice distiller's grains by papain was studied, which provided a basis for the development of antihypertensive functional food ingredients and the high-value utilization of liquor-making byproducts.