To achieve the high-value utilization of byproducts from the testes of Takifugu flavidus, we used testes of these fish as raw material and adopted enzymatic hydrolysis technology to prepare protamine peptides (TFPPs). Process parameters were optimized based on single-factor experiments combined with response surface methodology, and antioxidant activity and potential anti-fatigue mechanisms were systematically investigated. Papain was selected for enzymatic hydrolysis, for which the established optimal conditions were as follows: hydrolysis time, 4 h; temperature, 55 ℃; enzyme dosage, 10 000 U•g-1; and liquid-to-solid ratio, 15:1 (mL•g-1). Under these conditions, a 42.31% hydrolysis was achieved. Ultrafiltration membranes with different molecular weight cutoffs were used for fractionation and purification, resulting in three ultrafiltration fractions. In vitro antioxidant experiments revealed that the TFPP-1 fraction (<1 kDa) was characterized by the strongest antioxidant activity, with IC50 values of 8.94, 12.15, and 7.58 mg•mL-1 for the rates of DPPH, hydroxyl, and superoxide anion radical scavenging, respectively. Further identification of TFPP-1 using liquid chromatography–mass spectrometry/mass spectrometry yielded 2,085 peptide sequences. Key proteins associated with antioxidant and anti-fatigue activities, such as kelch-like ECH-associated protein 1 (Keap1), lactate dehydrogenase (LDH), and creatine kinase (CK), were selected as targets. Computer-aided virtual screening and molecular docking techniques were employed to examine the anti-fatigue peptides, with the results indicating that four peptides (DD6, DK6, FR6, and FD7) were characterized by strong binding to target proteins via hydrogen bonding and hydrophobic interactions; binding energies ranged from –9.5 to –6.7 kcal•mol-1, which are all less than –5 kcal•mol-1. It was hypothesized that these peptides have anti-fatigue effects by regulating the Keap1/Nrf2 antioxidant pathway, inhibiting LDH activity to reduce lactate accumulation, and modulating CK activity to optimize energy metabolism. In conclusion, the findings of this study will provide technical support and a theoretical basis for the high-value utilization of pufferfish testes and the development of testis-derived anti-fatigue peptides.