A new method to measure the three-dimensional refractive index distribution of optical fiber is proposed. A set of experiment system based on microscopic tomography with sample rotation is set up, which can quantitatively reconstruct the three-dimensional refractive index distribution inside optical fiber. The digital holograms are acquired at regularly-spaced angular positions within 180° viewing angle by the rotating sample in the digital holographic microscopy system of pre-amplification transmission type. Then, the refractive index integral projections for each degree′s optical fiber sample is calculated from the holograms. The three-dimensional refractive index distribution of the internal optical fiber is rebuilt by filtered back-projection algorithm. The experimental results of single-mode optical fiber and single-mode polarization maintaining optical fiber with the panda type demonstrate that the established system is feasible and effective in measuring the three-dimensional refractive index distribution of optical fiber.