As a member of EGFR family, ErbB3 is a critical heterodimerization partner of EGFR and ErbB2 in many EGFR- or ErbB2-driven cancers. As a ligand of ErbB3, heregulin β-1 (NRG1) induces the dimerization of ErbB3 with other ErbB family members and triggers downstream pathways. Using the directed protein evolution technique mRNA display, we successfully identified SDAHER3-A1 and SDAHER3-D5 that bind to ErbB3 with very high affinity and specificity in vitro and on cell surface. A biparatopic SDAHER3-D5A1 was constructed for improved biophysical properties and picomolar affinity (80.3 ± 20.3 pM) to ErbB3. SDAHER3-D5A1 binds to ErbB-expressing MCF7 cells with a binding affinity of 35.61 ± 17.07 nM. SDAHER3-D5A1 inhibits the NRG1-induced proliferation in a dose-dependent manner. SDAHER3-D5A1 is able to carry the conjugated AuNP to ErbB3-overexpressed cell and kill the cancer cell by hyperthermia treatment. Combined with Lapatinib, a kinase inhibitor, SDAHER3-D5A1 showed improved efficiency in inhibiting the proliferation of cancer cells with ErbB2 and ErbB3 overexpressed. An examination of downstream signaling pathways suggested that SDAHER3-D5A1 significantly decreased NRG1-induced ErbB3-/Akt-/ERK- phosphorylation and the combination with the drug further inhibits the phosphorylation. SDAHER3-D5A1-IRDye800 conjugates was synthesized and confirmed that SDAHER3-D5A1 can be used to target ErbB3-overexpressed tumor xenograft models and may be very useful for imaging purpose.