This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor (anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification. Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence experiments showed that increased expression of alpha-smooth muscle actin induced by TGF-beta 1 could be suppressed by this scFv antibody through inhibiting the phosphorylation of Akt.