The invention provides a method for separating an active ingredient, with an anti-oxidation effect, from human umbilical cord mesenchymal stem cell culture fluid. The active ingredient is prepared according to the following steps: in a process of normal cesarean delivery operation or eutocia, taking an umbilical cord under an aseptic condition, cleaning and disinfecting the umbilical cord, then removing blood vessels and mucosae, cutting umbilical cord tissues into pieces, and putting into a culture bottle for culture; taking the culture fluid of human umbilical cord mesenchymal stem cells growing in a log phase, and performing centrifugal ultrafiltration by adopting ultrafiltration centrifuge tubes with different specifications, thus finally obtaining an outlier having the anti-oxidationeffect and the molecular weight of 10 to 30Kda. The outlier can be freeze-dried into freeze-dried powder, thus facilitating storage and use. The human umbilical cord mesenchymal stem cell culture fluid outlier provided by the invention has good anti-oxidation activity, can help cells resist damage of free radicals; the outlier is from a human body tissue, is high in safety, and is expected to be applied to products such as drugs, health care products, skin care products and other daily chemicals. The separation method provided by the invention is simple and easy, and is low-cost, free of environment pollution and suitable for industrialized production.