Abstract: This study focused on oligopeptides derived from yellow chicken as the research object, aiming to investigate their antifatigue activity and potential functional peptide sequences. By establishing an antifatigue model through weighted swimming in mice, the mice were gavaged with low and high doses of yellow chicken oligopeptides for 28 consecutive days. An exhaustive swimming test was then conducted to assess the impact of yellow chicken oligopeptides on the swimming endurance of the mice after 26 days. Lactate dehydrogenase (LD), creatine kinase (CK) levels, and muscle/liver glycogen content changes in the mice were measured to evaluate their antifatigue effects in vivo after continuous gavage of mice for 28 days. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to analyze the peptide structure of yellow chicken oligopeptides, and Peptide Ranker was used for bioactivity prediction scoring. Furthermore, the solubility, toxicity, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of the peptides were screened using Innovagen, ToxinPred, and AdmetSAR online tools. Finally, molecular docking was performed using Discovery Studio 2019 Client software to identify potential antifatigue active peptides. The results showed that compared with the blank swimming control group, the weighted swimming times of the mice in the low and high dose groups of yellow chicken oligopeptides were significantly extended by 34.94% and 44.69%, respectively (P<0.05). Additionally, the oligopeptides obviously increased liver and muscle glycogen content while decreasing lactate dehydrogenase and creatine kinase levels, confirming their antifatigue efficacy. After screening through the bioactivity scoring system, 25 peptides with scores higher than 0.5 were obtained. Further activity analysis and molecular docking led to the identification of four peptides with good solubility, non-toxicity, excellent blood-brain barrier permeability, and intestinal absorption properties. These peptides tightly bind to the antifatigue receptor AMPA, specifically including (Gln-Pro-Arg, QPR), (Phe-Asp, FD), (Asn-Hyp, NX), and (Ala-Hyp, AX), with contents of 157.87, 4.40, 0.20, and 0.14 mg/100 g, respectively. These findings provide a solid material basis for the development of nutritional functional foods with yellow chicken oligopeptides to alleviate physical fatigue.