Molecular imaging, the visualization of molecular and cellular markers, is a promising method for detection of dysplasia and early cancer in the esophagus and can potentially be used to identify regions of interest for biopsy or tumor margins for resection. EGFR is a previously reported cell surface receptor with stepwise increases in expression during the progression from Barrett's metaplasia to adenocarcinoma. In this work, a 200 nm fluorescent nanoparticle contrast agent was synthesized for targeted imaging of EGFR through a series of surface modifications to dye-encapsulated polystyrene particles. Amino-functionalized polystyrene particles were PEGylated using a heterobifunctional PEG linker. Subsequently, thiolated M225 antibodies were conjugated to maleimide functional groups on attached PEGs for EGFR targeting. In vitro binding studies using flow cytometry demonstrated specific binding of M225-PEG-NP to EGFR-expressing cells with minimal nonspecific binding in EGFR(-) cells. Binding was shown to increase proportionally with the number of conjugated M225 antibodies. Adsorbed formulations with unmodified M225 antibodies, M225 + PEG-NP, were synthesized using the same antibody feeds used in M225-PEG-NP synthesis to determine the contribution of adsorbed antibodies to EGFR targeting. Adsorbed antibodies were less efficient at mediated nanoparticle targeting to EGFR than conjugated antibodies. Finally, M225-PEG-NP demonstrated binding to EGFR-expressing regions in human esophageal tissue sections.