Objective To construct a yeast-two hybrid system of DLX4 and identify its self-activation. Methods Human placenta yeast-two cDNA bank was constructed, and was identified by PCR. DLX4 bait plasmids including pDEST32-DLX4(4-1C), pDEST32-DLX4(4-1N) and pDEST32-DLX4(4-2) were constructed and transfected into yeast MaV203 cells, and the self-activation effect was detected. Results It was verified by PCR that human placental cDNA bank was successfully constructed. pDEST32-DLX4(4-2) and pDEST32-DLX4(4-1C) had no self-activation effect, and could be used in the yeast two-hybrid system. Conclusion Yeast-two hybrid bank and DLX4 bait plasmids have been successfully constructed, and can be used to screen the protein interacting with DLX4.