Abstract: To explore the potential mechanism of the NnWRKY40 transcription factor in lotus (Nelumbo nucifera Gaertn.) for regulating the synthesis of secondary metabolite alkaloids, a mixed cDNA library from different lotus tissues was constructed, and the proteins interacting with NnWRKY40 were screened. Total RNA of different tissues was extracted from ‘Baihuajian’, and a mixed cDNA library was established. The library capacity was 1.2 × 10⁷ CFU, recombinant rate was 100%, and average length of the inserted fragments was >1 000 bp. NnWRKY40 contains two homologous genes, NnWRKY40a and NnWRKY40b. As NnWRKY40b is reported to play a leading role in transcriptional activation of alkaloid synthesis genes, we used NnWRKY40b to construct the bait vector PGBKT7-NnWRKY40b. In total, 27 proteins interacting with NnWRKY40b were screened from the library using the co-transformation method. These interacting proteins could be divided into three categories: i.e., growth and development and stress response, hormone regulation and secondary metabolism, and unknown proteins. Six representative proteins, including NnUBC, NnPEBP, NnPPOA, NnCHS, NnJAZ1, and unknown protein 3, were selected for one-to-one verification, among which the JAZ protein was associated with alkaloid synthesis, suggesting that the NnWRKY40b transcription factor may be closely related to jasmonic acid (JA)-mediated regulation of alkaloid synthesis in N. nucifera.