Epidermal growth factor receptor (EGFR) is used as a biomarker for molecular imaging of cancer. Therefore, we synthesized two new Gd-DO3A-peptide complexes with an EGFR-binding peptide (NH2-CMYIEALDKYAC-COOH; EBP). To evaluate the effects of agents on targeting EGFR-overexpressing tumours, especially for more accurate cancer diagnosis and prognosis by magnetic resonance imaging (MRI), EBP-Gd-DO3A and EBP-(Gd-DO3A)3 were prepared. For in vitro cellular Gd accumulation and MRI analysis, after incubation with EBP-Gd-DO3A, EBP-(Gd-DO3A)3 or Gadovist® (a clinical macrocyclic agent) at equimolar Gd concentrations, the EGFR-overexpressing cells showed significantly higher cellular accumulation of the Gd-containing conjugates than the non-EGFR-overexpressing cells. While the cellular Gd accumulation with Gadovist® was nearly the same in all the cells. However, the cellular Gd accumulation with EBP-Gd-DO3A and EBP-(Gd-DO3A)3 was significantly reduced in the EGFR-overexpressing cells that were mocked with an inhibitory anti-EGFR monoclonal antibody, demonstrating the involvement of the EGFR pathway in the transport of both conjugates. In vitro MRI assays revealed stronger signal enhancement in the EGFR-overexpressing cells than in the non-EGFR-overexpressing cells. For in vivo pharmacokinetics and MRI analyses, the biodistribution and MR imaging was performed in the mice bearing MDA-MB-231 (EGFR-overexpressing) or U13MG (non-EGFR-overexpressing) tumour xenografts. The biodistribution and in vivo MRI experiments indicated that the improved target-specific signal enhancement of EBP-Gd-DO3A and EBP-(Gd-DO3A)3 was due to the increased accumulation of Gd in the EGFR-overexpressing tumour cells. The conjugates enabled EGFR-specific tumour MR imaging, demonstrating the potential clinical translation of these synthesized contrast agents for EGFR-based MR molecular imaging in vivo.