Objective To design, construct and express the Crossmab bispecific antibody molecular for targeting CD20 and HLA-DR. Methods The Crossmab bispecific antibody was designed, naming CD20-HLA-DR CrossmabCH1-CL, and two light chains and two heavy chains of the Crossmab were constructed by using genetic engineering technology. The constructed two heavy chain expression vectors and two light chain expression vectors were then co-transfected in FreeStyle 293-F cells and the Crossmab protein was expressed. Result The bispecific antibody protein moleculer CD20-HLA-DR CrossmabCH1-CL was obtained. Conclusion CD20-HLA-DR CrossmabCH1-CL targeting on CD20 and HLA-DR is successfully designed, constructed, expressed and characterized and laid the foundation for developing newly anti-tumor therapeutic bispecific antibody.