Multi-layer, tissue-like, multispectral enabled, three-dimensional tissue mimicking phantom devices are disclosed. The phantoms are targeted to be used with imaging modalities for calibration, optimization and standardization purposes. The manufacturing process results in a phantom with accurate geometry and realistic properties (e.g. optical). The above characteristics distinguish this approach from the currently available phantoms or manufacturing methods that neither have simple geometrical shapes (cubes, slabs, cylinders) nor use homogeneous approximations of the tissue properties and are usually calibrated for single wavelength tissue characterization. The embodiments described concern the fabrication of non-homogeneous phantoms with realistic anatomic geometry and optical properties matching the characteristics of any animal or human tissue type. Anatomical information is extracted from structural imaging modalities (MRI, XCT) or atlases and used to design digital phantoms. Prototypes are then manufactured by accurate 3D printing, which allows complex objects to be built layer by layer with sub-millimeter resolution. Then these prototypes are used to create negative molds with silicone that may be used for the finalized phantom. The final product may be constructed by loading the molds with the appropriate material (e.g. resin) with the desired tissue properties.