Objective:To prepare human herpesvirus 6 U94 protein and produce its antibodies. Methods:U94 gene of HHV6 was amplified by PCR and sequenced. The resulting DNA construct was cloned into a prokaryotic expression vector(PGEX-6P-1). The recombinant plasmid was transformed into Eserichia coli Rosetta. The accuracy of inserted gene and specificity of proteins were detected by two enzymes digestion, SDS-PAGE, and Western Blot. The fusion U94 protein purified by affinity chromatograph was used to vaccinate rabbit to produce antibodies. Results:The purity of U94 protein was above 90%. The titer of the antibodies was 1 ∶ 100 000. Conclusion:HHV6 U94 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.