Abstract: The aim of this study was to purify angiotensin I-converting enzyme (ACE) inhibitory glycopeptide from quinoa fermented by Mucor wutungkiao and Rhizopus oryzae. The fermentation process conditions were optimized by a single factor experiment and a response surface experiment. The fermentation liquid was separated and purified by vacuum concentration, alcohol precipitation, Sephadex G-15 and reverse-phase high performance liquid chromatography. Fourier infrared spectroscopy was used to determine the composition of functional groups and β-elimination method to determine the glycopeptide bonding type. The effects of temperature, pH, metal ions, and in vitro simulated digestion on the activity were analyzed. The results showed that the optimal fermentation parameters were as follows: 8.7 d of fermentation time, 2.8% (φ) of total inoculum size containing Mucor wutungkiao to Rhizopus oryzae in a volume ratio of 1:1, and 1:18 g/mL of a solid-liquid ratio. The ACE inhibition rate was 64.22%±4.57%. A novel glycopeptides F₃b, was isolated and purified by ethanol precipitation, Sephadex G-15, and reverse-phase high performance liquid chromatography. Fourier-transform infrared spectroscopy proved the presence of polypeptides and polysaccharides in F₃b, and β-elimination reaction further demonstrated that the glycoprotein was an O-linked type. In vitro stability results indicate that the ACE inhibitory activity of glycopeptides was hardly changed under heating conditions from 25 to 55 ℃, and was 86.23%±3.47% of the original activity at 100 °C. Conversely, pH (from 2 to 12) had no significant effect (P>0.05) on the ACE inhibitory activity of glycopeptides. 4 mmol/L of Zn²⁺ and Fe³⁺ increased the ACE inhibitory activity of F₃b to 109.91%±8.12% and 117.43%±6.78% of the control, respectively. While same concentration of K⁺ and Ca²⁺ reduced the ACE inhibitory activity to 78.94%±2.18% and 85.31%±4.99% of the control, respectively. After simulated gastrointestinal digestion, the ACE inhibitory activity of F₃b decreased to 70.00%±3.30% of the control. In summary, this study could enrich the variety of ACE inhibitory peptides and provide technical reference for high-value utilization of quinoa.