The invention discloses a preparation method of Miltenberger blood group series phenotype red blood cells. The preparation method includes the following steps: step one, adopting monoclonal antibodiesto identify common clinically significant Rh, Kidd, MNS, Duffy, Lewis and P system antigens on Miltenberger blood group positive type O red blood cells; step two, respectively performing PCR-SSP andgene sequencing validation on dose effect antigen C, c, E, e, M, N, S, s, Fya, Fyb, Jka and Jkb to obtain a red blood cell surface antigen pattern; step three, using a red blood cell reagent preservation solution to prepare into a Miltenberger blood group series phenotype red blood cell reagent; and step four, using the prepared Miltenberger blood group series phenotype red blood cell reagent andan existing irregular antibody screening cell reagent to parallelly detect the serum of a hospitalized blood transfusion patient, vertifying the diagnostic performance of the cell reagent, and obtaining a final product. Through the cooperation of the prepared Miltenberger blood group series phenotype red blood cells and the existing screening cells, detection can be performed on Miltenberger bloodgroup related antibodies, and therefore, safe blood transfusion can be guaranteed.