This study used locally sourced black, red, and white quinoa from Zhangjiakou as experimental materials to optimize the preparation process of quinoa antioxidant peptides and identify their sequence characteristics. Protein was extracted using the alkali-soluble acid precipitation method. The optimal hydrolytic enzyme was selected by comparing the hydrolysis effects of eight proteases. Enzymatic hydrolysis conditions were optimized using single-factor experiments and response surface methodology. After purification by Sephadex G-15 gel filtration chromatography, peptide sequences were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) combined with De novo sequencing technology. The results showed that black quinoa and alcalase were identified as the optimal raw material and hydrolytic enzyme, respectively. The optimal enzymatic hydrolysis conditions were an enzyme dosage of 4000 U·g⁻¹, hydrolysis time of 3.5 h, temperature of 42 °C, and pH 7.6, under which the average DPPH radical scavenging rate reached 86.61%. The purified G-1 fraction exhibited the highest antioxidant activity. A total of 1549 peptide sequences with confidence ≥90% were identified, among which peptides containing two or more hydrophobic amino acids accounted for 60.62%. This compositional feature is consistent with structural characteristics commonly associated with antioxidant peptides. This study provides a reference for the processing of quinoa-based functional ingredients.