We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed “hot-spot” locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2 mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.