The invention discloses a method for separating fetal nucleated red blood cells in maternal peripheral blood. The method comprises the following steps: (a) enriching nucleated cells, namely, (1) collecting whole blood, (2) centrifuging, and collecting cell precipitates, (3) preparing cell suspension, namely adding a cell culture medium to the cell precipitates, and uniformly mixing so as to prepare the dilute cell suspension, (4) putting a cell separating medium in a centrifugal tube in advance, and adding the dilute cell suspension to a layering liquid surface, (5) centrifuging so as to obtain a cell layer, and (6) sucking up the cell layer, adding Hank, s liquid, uniformly mixing, centrifuging at a room temperature, and washing cells, and (7) resuspending the cells; and (b) screening the fetal nucleated cells in the maternal blood, to be specific, (9) separating out the fetal nucleated red cells, and (10) carrying out quadruple morphology identification on nucleated red cell pictures obtained by sorting, and collecting the nucleated red cells meeting the standards for extracting the fetal whole genome DNA (deoxyribonucleic acid). According to the method, the purity and the recovery rate of the fetal nucleated red blood cells obtained by the separation and purification of the maternal peripheral blood are high.