A yeast two-hybrid cDNA library of penultimate leaf harvested from the rice cultivar Zhonghua11 before its flowering transition was constructed to study the molecular network of flowering regulation.The high-quality total RNA was isolated and the first-strand cDNA was obtained by reverse transcription using primer Oligo(dT).The double-strand cDNA was amplified by using Long Distance PCR.The cDNA library of rice penultimate leaf was generated by using homologous recombination-mediated SMART technology in yeast strain AH109.The testing results of library’s quality showed that the transformation efficiency of cDNA library was 1.185×10 6 transformants/3 μg pGADT7-Rec with the titers of 2.65×10 8 cfu/mL and the recombinant rate of 94.7％.The sizes of inserted fragments were ranged from 350 bp to 2 000 bp.These data indicated that the cDNA library could be used to screen interacting proteins of the flowering time gene in rice.