Abstract: In order to realize non-contact and high-precision measurement of the radial run-out of the spindle in the machining state, the laser Doppler differential detection method based on the optical heterodyne method was used for theoretical analysis and experimental verification. The application of direct coupling and lens-coupling all-fiber optical path reduces the adjustment difficulty of the Bragg diffraction optical path, improves the measurement resolution and anti-interference ability, and reduces the noise; the optical fiber splitter realizes the differential measurement on the same axis, suppressing interference caused by the vibration of the measuring optical path itself. The radial run-out measurement result of the spindle with a relative error of 0.0838% is obtained, and the nanometer run-out error measurement is realized. The results show that the relative error and measurement uncertainty of the system are less than 0.1%. This study has a certain guiding significance for the real-time measurement of the radial run-out of the main shafts.