Superparamagnetic iron oxide nanoparticles (SPIONs) have gained attention as contrast agents in cancer imaging due to their unique magnetic properties, enhancing MRI's effectiveness. This study introduces an innovative approach by functionalizing SPIONs with diethylenetriaminepentaacetic acid (DTPA) and a novel C-peptide derived from endostatin, aimed at improved tumor targeting. This C-peptide targets integrin αv receptors, prominently overexpressed in breast cancer cells, enhancing specificity and imaging efficacy. The SPION-DTPA-C-peptide provided precise MRI capabilities and significantly inhibited cell viability and migration in vitro (p < 0.01). The DTPA coating also facilitates the chelation of technetium-99m (Tc), allowing dual-modality imaging with SPECT. Comprehensive characterization via XRD, EDX, TEM, FT-IR, and VSM confirmed successful synthesis, functionalization, spherical morphology, optimal size, and superparamagnetic characteristics. In vitro studies demonstrated selective targeting of 4T1 mammary carcinoma cells by SPION-DTPA-C-Peptide, exerting cytotoxic effects and inhibiting cell migration. In vivo imaging in Balb-c mice bearing 4T1 xenograft tumors showed enhanced tumor targeting and contrast on both MRI and SPECT modalities. These findings highlight the potential of the SPION-DTPA-C-Peptide system for targeted cancer imaging, offering a promising strategy for integrated MRI and SPECT in cancer diagnosis and management.