As a novel member of the IAP family, survivin was observed to express in the most common human cancers. Anti-cancer therapy targeting survivin has drawn considerable attention. This report presented firstly construction of recombinant plasmid pRSET-B-TAT-survivin (T34A), expression in Escherichia coli, purification, renaturation, and bioactivity. The cDNA encoding survivin was cloned by RT-PCR from breast cancer cell lines B-cap37. Expression vector pRSET-B-TAT-survivin (T34A) was constructed by PCR after survivin was mutated by PCR site-directed mutagenesis. Recombinant TAT-survivin (T34A) protein was expressed highly in E. coli BL21 (DE3) by 0.5 mM IPTG induction and its yield could reach 650 mg/l in fermentation culture. The fusion protein in a form of inclusion body was then solubilized, refolded, and purified to a purity of 98% by cation exchange chromatography and size-exclusion chromatography. Four hundred and eighty milligrams protein of interest was obtained in per liter fermentation culture. The protein of interest was identified by SDS-PAGE and Western blot analysis, and great bioactivity of target protein to two cancer cell lines was confirmed by morphological changes and evaluated by MTT. The findings suggested that recombinant protein TAT-survivin (T34A) has a bright future in cancer therapy targeting towards survivin, and the efficient procedure of expression and purification may be useful for the mass production of this therapeutically important protein.