Using pET-30a and pEGX as starting vectors, the non-fusion expression plasmids pET-BlfFf and the fusion expression plasmids pEGX-BlfFf were constructed for preparation of bovine lactoferrin functional fragment(BlfFf) and the protein expression was induced in Escherichia coli BL21. SDS-PAGE analysis showed that the target protein expressed from pET-BlfFf is mainly in the form of inclusion bodies, while the target protein expressed with pEGX-BlfFf could be dissolved in a large number of cell-shredding supernatants. This indicates that the pEGX-BlfFf is more applicable for soluble BlfFf production. The pEGX-BlfFf expression product was purified by GST label affinity column chromatography, and the soluble protein GST-BlfFf was purified. The yield was about 60 mg/L. The target protein BlfFf was finally purified after remove its GST by treated with thrombin. The bioactivity analysis of BlfFf showed that the peptides from BlfFf hydrolyzed by pepsin shows antibacterial activity against both Staphylococus aureus and Escherichia coli.