AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation,digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture.METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation.cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase Ⅲ in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2000 to test their inhibition efficiency.RESULTS: siRNAs corresponding to part of the hepatitis3 virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP.CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.