In the experiment, the preparation technology of the high Fischer ratio oligo-peptide (HFP) from Trichiurus japonicus (HFP-T) was studied, and the parameters on two-step sequential enzymolysis and removing aromatic amino acids (AAA) using activated carbon were optimized. The F value of the HFP was confirmed by determination of amino acid components. Taking hydrolysis degree and protein conversion ratio as criterions, binary-enzymes hydrolysis process was optimized and the results were as follows: pepsin was selected as the first enzyme, and its optimal parameters were ratio of enzyme to substrate 3%, ratio of meat to water 1∶5 (g/mL), temperature 35 ℃, pH 2.0 and enzymolysis time 3 h; flavourzyme was selected as the second enzyme, and its optimal parameters were ratio of enzyme to substrate 3%, pH 7.0, temperature 50 ℃ and enzymolysis time 3h. The highest hydrolysis degree and F value (OD₂₂₀/OD₂₈₀) of hydrolysate prepared under the optimal parameters were 40.51%±3.18% and 5.75, respectively. In order to raise the F value of the sample by removal of AAA, five kinds of activated carbons including one powdered and four granular activated carbons were chosen to evaluate their removing AAA effects, and the results indicated that No.4 activated carbon (200 mesh) have the best removing AAA effects under the parameters of solid-liquid ratio 1∶20 (g/mL) and pH 6.0 for 4 h at room temperature, and the F value (OD₂₂₀/OD₂₈₀) of hydrolysate reached 30.72 from 5.75. The F value (BCAA/AAA) of hydrolysates (removing AAA) measured on Automatic Amino Acid Analyzer was calculated to be 28.06, and the content of Lysine was 22.403 μmol/L. The results of activity tests showed that HFP-T can significantly prolong time of the loss of righting reflex and shorten time to sober up, increase liver and muscle glucogen contents and lower blood urea nitrogen and blood lactate levels after mice have exercised. HFP-T also enhanced the activity of lactic dehydrogenase to scavenge excessive LA for slowing the development of fatigue. In addition, HFP-T increased the activities of superoxide dismutase, catalase and glutathione peroxidase to reduce the reactive oxygen species (ROS) damage in mice. In conclusion, HFP-T possessed good anti-fatigue capacities in mice by inhibiting the oxidative reactions and provided an important basis for developing the swim bladder peptide functional food.