Objective：To prepare humanized antinuclear antibody Fab fragment. Methods: The reconstructed humanized antinuclear antibody (ANA) Fab phage display library was enriched by 4 rounds of panning and was identified by indirect ELISA method.Phasmid DNA isolated from positive clones was deprived of gⅢ gene. After self-ligation the recombinant plasmid was used to transform E.coli. XL1-Blue, then XL1-Blue was induced by IPTG to product soluble human antinuclear antibody Fab fragment. Finally, soluble human antinuclear antibody Fab in the supernatant was identified by indirect ELISA method and immunofluorescence. Results: The eluted phages were enriched by more than 200 folds after 4 rounds of panning. Two positive clones were isolated from the ANA Fab library. Electrophoresis after XhoⅠdigestion proved that the self-ligation was successful after deletion of gⅢ gene. The results of indirect ELISA indicated that the 2 positive clones of Fab had specific anti-dsDNA activity. Indirect immunofluorescence showed homogeneous fluorescence within nuclei of Hep2 and monkey hepatic cells and in the Crithidia kinetoplast. Conclusion: We have successfully prepared soluble, specific human antinuclear antibody Fab fragment, which paves a way for preparation of high affinity antinuclear antibody Fab fragment.