This study aims to screen anti-exercise fatigue peptides from enzymatic hydrolysates of Cervus nippon blood. The anti-exercise fatigue peptides were analyzed by swimming time (ST) under load, blood urea nitrogen (BUN) content, blood lactic acid (BLA) and hepatic glycogen (HG) content of the mice. Dialysis and Sephadex G-25 dextran gel chromatography were applied for isolation and purification. The contents of free amino acid and hydrolyzed amino acid were detected by automatic amino acid analyzer. The contents of γ-aminobutyric acid (GABA) and taurine were determined by high performance liquid chromatography. Gel permeation chromatography was utilized for the molecular weight distribution and liquid chromatography-tandem mass spectrometry was utilized for the identification of peptides. Dialysis produced three fractions: molecular weights of 500-3 500 Da (M-1), 3 500-8 000 Da (M-2) and > 8 000 Da (M-3) and Sephadex G-25 gel chromatography produced two fractions from M-1: P-1 and P-2. Compared with the normal saline group, M-1 significantly (P＜0.01) increased the ST under load, the HG content and decreased the BUN content, the BLA content in mice, P-2 could significantly (P＜0.05) increase the ST under load and decrease the BUN content, the BLA content in mice. The content of leucine from M-1 was the highest (12.14%). The content of aspartate from P-2 was the highest (13.54%). Compared with P-2, the content of GABA and taurine from M-1 were increased by 178.39% and 51.04% respectively. M-1 composed of peptides with amino acids less than 20 and molecular weight less than 2 000 Da. M-1 were screened from the enzymatic hydrolysates of Cervus nippon blood. The results of this study could provide a theoretical basis for the development of anti-exercise fatigue products from Cervus nippon blood.