During spermatogenesis of most animals, the basic proteins associated with DNA are continuously changing: somatic-typed histones are partly replaced by spermatoza-specific histones, which are then replaced by transition proteins, which in turn are finally replaced by protamines. With replacement of sperm basic nuclear proteins (SBNP), nuclei progressively undergo condensation. The nuclei of spermatozoa in Chinese crab Eriocheir sinensis (Decapoda, Crustacea) are found to be uncondensed. However, the mechanism of nuclear uncondensation is not clear. To study histone H3 dynamics during E. sinensis spermatogenesis, H3 coding sequence of E. sinensis was amplified by PCR and cloned into prokaryotic expression vector pET-30a(+), which was then transferred into Escherichia coli Rosetta (DE3) and induced to express the recombinant His/H3 protein by 0.2mmol/L isopropyl β-D-thiogalactoside (IPTG). Meanwhile, the recombinant protein was purified through Ni-NTA Agarose affinity chromatography and purified protein was used as antigen to inoculate Oryctolagus cuniculus to produce antiserum. The polyclonal antibody was tested using western blotting method. Finally, H3 gene coding region from E. sinensis was cloned, prokaryotic expression vector pET-30a-H3 was constructed. SDS-PAGE showed that a 21 ku protein was expressed successfully in Rosetta (DE3), and the recombinant protein existed in the form of supernatant and inclusion body, and recombinant protein was purified well through Ni-NTA Agarose affinity chromatography. Western blot showed that the polyclonal antibody had high specificity. In summary, coding sequence of histone H3 in E.sinensis was successfully cloned, prokaryotic expression vector pET-30a-H3 was constructed and recombinant protein was purified. The polyclonal antibody with high specificity was produced, which might provide fundamental basis for further study of histone H3 dynamic changes during E. sinensis spermatogenesis.