The invention discloses a three-cistron expression vector, a preparation method and application and belongs to the technical field of gene engineering.The three-cistron expression vector comprises a nuclear matrix combining region sequence and a three-cistron sequence which is formed in the mode that two internal ribosomes enter a site sequence and connected; the structure of the three-cistron sequence is promoter-light chain signal peptide SPL-IRESwt-heavy chain signal peptide SPH-IRESatt-selection marker-Poly A.The vector can express a light chain, a heavy chain and a selection marker gene of an antibody at the same time, the problem that expression of a heavy chain, a light chain and a selection marker gene of a traditional vector is not balanced is solved, antibody quality is improved, and the cloned screening rate of positive cells is raised; meanwhile the nuclear matrix combining region sequence contained on the vector can further overcome transgene silencing, efficient and long-term expression of transgenes in host cells is achieved, on one hand, the antibody protein expression level is improved, and on the other hand, efficiency of screening of subsequent monoclonal antibody cell lines is improved.