Abstract: Objective Triple negative breast cancer(TNBC) represents a group of refractory breast cancers with aggressive clinical manifestations as well as poor prognoses. Human epidermal growth factor receptor(EGFR) expression is strongly associated with TNBC progression and it may serve as a therapeutic target for TNBC. We aimed to evaluate EGFR affibody-based PET imaging to profile EGFR expression in small animal models. Methods 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) conjugated Ac-Cys-Z_EGFR:1907 was chemically synthesized using solid phase peptide synthesizer and then radiolabeled with ⁶⁴Cu. The in vitro cell uptake study was performed using SUM159 and MCF7 cells. The biodistribution and small animal PET imaging using ⁶⁴Cu-DOTA-Z_EGFR:1907 were further carried out with nude mice bearing subcutaneous MDA-MB-231 and SUM159 tumors. Results DOTA-Ac-Cys-Z_EGFR:1907 was successfully synthesized and radiolabeled with ⁶⁴Cu. Biodistribution study showed that tumor uptake value of ⁶⁴Cu-DOTA-Ac-Cys-Z_EGFR:1907 remained at(4.07?.93)%ID/g at 24 h in nude mice(n=4) bearing SUM159 xenografts. Furthermore, small animal PET imaging study clearly showed that ⁶⁴Cu-DOTA-Ac-Cys-Z_EGFR:1907 specifically delineated the EGFR positive TNBC tumors at 4 h or later. Conclusion The study demonstrates that ⁶⁴Cu-DOTA-Ac-Cys-Z_EGFR:1907 is a promising molecular probe for PET imaging of EGFR positive TNBC. EGFR based small protein scaffold holds great promise as a novel platform that can be used for EGFR profiling of TNBC.