Abstract: Although tuna blood is a nutrient-rich processing byproduct, its utilization poses significant challenges. This study aimed to promote the high-value utilization of tuna by-products by evaluating the antioxidant properties of enzymatic hydrolysate-derived tuna blood peptides (TBPs) to facilitate their industrial application. We used DPPH and ABTS radical scavenging abilities as indicators to analyze their antioxidant capacity under various conditions, including different temperatures, pH values, metal ions, and in vitro gastrointestinal simulations. We employed gel filtration and ultrafiltration to separate and purify the tuna blood peptides, and studied the antioxidant activities of the four resulting fractions. The results show that the peptides exhibited good antioxidant capacity at 0–20 °C, under acidic conditions, and in the presence of certain metal ions. In the simulated gastrointestinal digestion, the DPPH radical scavenging ability remained at a high level after digestion with pepsin and trypsin, while the ABTS radical scavenging ability decreased significantly. We obtained four fractions through separation and purification, with the F4 fraction (Molecular mass<1 000 Da) exhibiting the strongest antioxidant activity. The study shows that purified low-molecular-mass blood peptides (<1 000 Da) exhibit higher antioxidant activity, and the antioxidant performance of tuna blood peptides is jointly affected by environmental conditions and molecular mass.