Abstract： Objective To purify human 14-3-3β (YWHAB) recombinant protein expressed in the E.coli, prepare its antiserum and construct the eukaryotic expression vector for transfecting mammalian cells. Methods The human 14-3-313 recombinant protein expression vector pET30a (+) /YWHAB constructed by the ORF of YWHAB gene and prokaryotic expression vector pET30a (+) was transformed into E.coli BL21 (DE3). The expression of the recombinant protein was induced by IPTG and the protein was purified by affinity chromatography on a Ni-NTA resin. BALB/c mice were immunized by the purified protein, and ELISA and Western blotting were employed to detect the titer and specificity of the antiserum. The open reading flame of YWHAB gene was obtained by PCR, the purified PCR product digested by BamH Ⅰ and EcoR Ⅰ was cloned into the eukaryotic expression vector pEGFP-N1, and the product digested by BamH Ⅰ and Hind Ⅲ was cloned into the eukaryotic expression vector pCDNA3.1 (+). The recombinant vectors were identified by PCR and enzyme digestion. Results The recombinant protein was expressed as a soluble protein with a relative molecular mass of about 32 kD, which was consistent with the expected value. The recombinant protein was purified using affinity chromatography to yield a purity up to 90%. The antiserum had high specificity and titer (1: 50000). The results of PCR and enzyme digestion verified successful construction of the eukaryotic recombinant expression vector pEGFP-N1/YWHAB and pCDNA3.1 (+)/YWHAB. Conclusion The recombinant human 14-3-3β protein, the antiserum and the eukaryotic expression vector obtained may facilitate further functional study in the human 14-3-3β protein.