To achieve fast and accurate three-dimensional measurements of microspheres in liquid, especially axial tracking resolution and speed, this paper proposes a new measurement method based on off-focus measuring theory and combining quadrant interpolation algorithm and radial projection algorithm. Resolution of 1 nm measurement in 3D directions of microspheres is realized. The method has high measurement efficiency with the fastest measuring rate at individual particle axial position of hundred frames per second and more. The influences of horizontal deviations on axial measurements are further discussed, and axial resolution throughout dozens of micrometers is analyzed. Compared with the cross-correlation method under the same experimental conditions, this measurement method shows feasibility in practical measurement applications and enjoys higher measurement efficiency when achieving the same axial measurement resolution.