The mature nodules of Medicago truncatula (A17) were used to construct a cDNA library with Gibson cloning technique and SMART cDNA library construction technique.The result of library testing showed that the transformation efficiency of the library was 7.74×105 transformants/3 μg pGADT7-Rec-New.Some colonies were randomly picked to run PCR.Results showed that the inserted DNA fragment size was ranged from 0.5 kb to 2.0 kb,with the recombinant rate of 91.7%.The cDNA library was stored as plasmid.This method can be used to construct library for yeast one or double hybrid and other plasmid library.The library was used to find protein(TF) that can interact with the DNA fragment of interest and some TFs were obtained.