Ex vivo diffusion tensor imaging (DTI) are widely used in experimental studies for excellent images. The comparison between in vivo and ex vivo DTI has been conducted without the same scan parameters. We aimed to compare the living and fixed white and gray matters under the same condition and explore the effects of coil and signal average. Diffusivities were significant different between living and fixed brains. Coil and signal average significantly affected the signal-to-noise ratio of ex vivo white and gray matters. The results indicate that fixation and MR conditions should be considered in clinical and experimental DTI studies.