The organization of flavonoids biosynthesis metabolon is varied in different plant species.Although there have been reports of flavonoid metabolic compartments in many plants, research on flavonoid metabolon in mulberry trees has not been reported yet. CHI-fold proteins are important components of most reported flavonoid metabolons. Therefore, understanding the function and interaction network of CHI-fold protein is one of the important keys to deciphering the mechanism of flavonoid biosynthesis. Based on current research findings, we believe that there may be more complex flavonoid metabolon and CHI-fold based regulation of flavonoids biosynthesis pathway in mulberry trees. In the present study, we identified four CHI-fold proteins including CHI1, CHI2N, CHI2D and CHIL. CHIL is the orthology of the arabidopsis EFP and CHI2-D is the production of the alterative transcription of CHI2which contained 37 amino acids truncated at the N-terminus. CHI2N is corresponding to CHI2. The difference in catalytic efficiency for MaCHI2N, MaCHI2D and MaCHI1 against NC and their expression levels intrinsically provided a complicated regulation of flavonoids influx.Only MaCHIL was found obvious interaction with MaCHS1 and considered as component of flavonoid metabolon in mulberry. In addition, the ligands NC and NA also affected the CHIL-CHS interaction and CHILs were thought to act as sensors for ligand-mediated pathway feedback by influencing CHS function.