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Three-dimensional and three-component (3D3C) velocity measurements have long been desired to resolve the 3D spatial structures of turbulent flows. Recent advancements have demonstrated tomographic particle image velocimetry (tomo-PIV) as a powerful technique to enable such measurements. The existing tomo-PIV technique obtains 3D3C velocity field by cross-correlating two frames of 3D tomographic reconstructions of the seeding particles. A most important issue in 3D3C velocity measurement involves uncertainty, as the derivatives of the measurements are usually of ultimate interest and uncertainties are amplified when calculating derivatives. To reduce the uncertainties of 3D3C velocity measurements, this work developed a regularized tomo-PIV method. The new method was demonstrated to enhance accuracy significantly by incorporating the conservation of mass into the tomo-PIV process. The new method was demonstrated and validated both experimentally and numerically. The results illustrated that the new method was able to enhance the accuracy of 3D3C velocity measurements by 40%–50% in terms of velocity magnitude and by 0.6°–1.1° in terms of velocity orientation, compared to the existing tomo-PIV technique. These improvements brought about by the new method are expected to expand the application of tomo-PIV techniques when accuracy and quantitative 3D flow properties are required.