Objective To  screen hepatocellular candidate binding proteins  interacting with pre-X fusion protein (pre-X) of hepatitis B virus by yeast-two hybrid technique. Methods Polymerase chain reaction (PCR) method was applied  to amplify pre-X gene. The  targeted gene of pre-X was cloned  into yeast expression plasmid pDEST32  to construct bait plasmid pDEST32-pre-X. Western blot method was employed  to  test pre-X expression after pDEST32-pre-X being  transformed  into  the yeast cell MaV203 by LiAc-mediated method. Both pDEST32-pre-X and pDEST22-cDNA were contemporarily  transformed  into MaV203  to screen the binding protein of pre-X. MaV203 was plated on synthetic dropout nutrient medium (SC/-Trp-Leu-His-Ura) and containing X-gal  for selection and screening. After  that,  the prey plasmids  from  true positive colonies were extracted and  sequenced. The partial cDNA  sequence  in prey plasmids were analyzed by bioinformatics software. Results The yeast expressed vector pDEST32-pre-X was successfully constructed. After screening, 3 pieces of cDNA  in prey plasmids  from  true positive blue colonies were sequenced. The cDNA sequence was a TCP1 protein. Conclusions Yeast-two hybrid method was successfully applied  for screening TCP1 protein as candidate binding protein of HBV pre-X.