We have investigated the potential of tissue phantoms fabricated with thermosoftening- and photopolymerization-based three-dimensional (3D) printers for use in evaluation of biophotonic imaging systems. The optical properties of printed polymer samples were measured and compared to biological tissues. Phantoms with subsurface channels as small as 0.2 mm in diameter were fabricated and imaged with microscopy, x-ray microtomography, and optical coherence tomography to characterize morphology. These phantoms were then implemented to evaluate the penetration depth of a hyperspectral reflectance imaging system used in conjunction with a near-infrared contrast agent. Results indicated that 3D printing may provide a suitable platform for performance testing in biophotonics, although subsurface imaging is critical to mitigate printer-to-printer variability in matrix homogeneity and feature microstructure.