Purpose The aim was to develop a method for reproducible orbital volume (OV) measurement in vivo based on 3D printing. Methods Twelve orbits were obtained from dry skulls of the Human Anatomy Department of Lille University. Computer tomography (CT) slice images of these orbits were transformed into stereo-lithography (STL) format and 3D-printed. Bone openings were closed using either putty and cellophane after printing (3D-Orb-1) or at the printing stage in silico using MeshMixer (3D-Orb-2). The results were compared with those of the conventional water-filling method as a control group (Anat-Orb). Results The observers reported a mean orbital volume of 21.3 ± 2.1 cm3 for the open-skull method, 21.2 ± 2.4 cm3 for the non-sealed 3D-printing method, and 22.2 ± 2.0 cm3 for the closed-print method. Furthermore, the intraclass correlation coefficients (ICCs) showed excellent intra-rater agreement, i.e., an ICC of 0.994 for the first observer and 0.998 for the second, and excellent interobserver agreement (ICC: 0.969). The control and 3D-Orb-1 groups show excellent agreement (ICC: 0.972). The 3D-Orb-2 exhibits moderate agreement (ICC: 0.855) with the control and appears to overestimate orbital volume slightly. Conclusion Our 3D-printing method provides a standardized and reproducible method for the measurement of orbital volume.