Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single-chain anti-EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR-expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR-QDs or ScFvEGFR-IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular-targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR-targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.