Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps including Butyl-Sepharose FF, DEAE-Sepharose FF, 1% Triton X-114 phase separation and Sephadex G-25. Results showed that HSP65-Her2 was purified up to 97% purity and was able to generate Her2-specific cytotoxic T lymphocytes (CTLs), suggesting that the scheme is efficient for purifying the non-tagged HSP65-Her2 fusion protein with biological activity.