This work presents a comprehensive methodology for constructing a tissue equivalent mouse phantom using image modeling and 3D printing technology. The phantom can be used in multimodality imaging and irradiation experiments, quality control, and management. Computed tomography (CT) images of a mouse were acquired and imported into 3D modeling software. A skeleton and skin shell models were segmented in the modeling software and manufactured using 3D printing technology. The bone model was constructed with VERO-WHITE printing material with additional ingredients, including a photosensitive resin, polyurethane epoxy resin, and acrylate. Acrylonitrile butadiene styrene resin material was used to construct the skin shell. The skin shell was attached to the skeleton and filled with a specially formulated gel to act as a soft tissue substitute. The gel consisted of agarose, micro-pearl powder, sodium chloride, and magnevist solution (gadopentetate dimeglumine). A micro-container filled with 18F-fluorodeoxyglucose (18F-FDG) radioactive tracer was placed in the abdomen for micro and human positron emission tomography (PET)/CT imaging. The mouse phantom had tissue equivalency in dose attenuation with x-rays and relaxation times with magnetic resonance imaging (MRI). The CT Hounsfield Unit (HU) range for the gel soft tissue material was 31-36 HU. The 3D printed bone mimetic material had equivalent tissue/bone contrast compared with in vivo mouse measurements with a mean value of 130 ± 10 HU. At different magnetic field strengths, the T 1 relaxation time of the soft tissue was 382.75-506.48 ms, and T 2 was 51.11-70.76 ms. 18F-FDG tracer could be clearly observed in PET imaging. The 3D printed mouse phantom was successfully constructed with tissue-equivalent materials. Our model can be used for CT, MRI, and PET as a standard device for small-animal imaging and quality control.