The present invention is preparation process of active black-bone chicken peptide. By using black-bone chicken as material and through advanced enzymolysis and membrane separation process, active peptide with high peptide content and molecular weight 6000 Da is prepared. The active peptide is easy to be absorbed by human body, has the bioactivities of antagonizing fatigue, nourishing blood, raising immunity, etc, and is especially suitable for nourishing and treating of persons with different deficiencies. The preparation process is reasonable and environment friendly, has normal temperature separation, low power consumption, simple operation and high efficiency, and is suitable for industrial production.