Provided is a pegylated erythropoietin conjugate having the general formula of rhuEPO-NH-CH2-X-S-Y-mPEG. Also provided is a preparation method of the conjugate comprising carrying out reductive amination on erythropoietin and aldehyde compound containing protected sulfhydryl to form activated erythropoietin linked to -NH-CH2- bond, then carrying out methoxypolyethylene glycol derivative coupling reaction, thus obtaining the conjugate. The conjugate or pharmaceutical composition comprising the conjugate can be used for the treatment of diseases involving erythropoietin insufficiency or red blood cell insufficiency or defect.
