Human epidermal growth factor receptor 2 (HER2)is a powerfultarget for cancerimmunetherapy.The development of anti-HER2 monoclonal antibodiestargeting different domains of HER2 is quiteeffective. However, the selection and production of multivalentantibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificiallibrary by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2melanoma cellsand HER2-nagative cells. DARPin selected from the library could be expressed in solubleform with a yieldof 70 mg/L.After purifiedby two continuousand easy steps, the purity of DARPin was 90% as established by SDS-PAGE and RP-HPLC.Selected DARPin showedsignificant HER2-targeting abilitywith an affinity of 1.05± 0.47μM. MTT assay demonstrated that at the concentration of 640 nM, the selected DARPin dimercould inhibit the SK-BR-3 growth at a rate of36.63%and 46.34%in 48 h and 72 h incubationseparately, which was similar to trastuzumab(43.12%and Downloaded by [University of New England] at 00:44 14 January 2018 449.14%separately). These findings suggestedthatit was an effective methodto select antibody mimeticDARPinby bacteria surface displaycombined with live cellssortingandprovideda drug candidate for cancer therapy