Some studies have reported that scaffold or cell-based transplantation may improve functional recovery following SCI, but no imaging information regarding regeneration has been provided to date. This study used tractography to show the regenerating process induced by a new biomaterial-aligned fibrin hydrogel (AFG). A total of eight canines subjected to SCI procedures were assigned to the control or the AFG group. AFG was implanted into the SCI lesion immediately after injury in 5 canines. A follow-up was performed at 12 weeks to evaluate the therapeutic effect including the hindlimb functional recovery, anisotropy and continuity of fibers on tractography. Using tractography, we found new fibers running across the SCI in three canines of the AFG group. Further histological examination confirmed limited glial scarring and regenerated nerve fibers in the lesions. Moreover, Repeated Measures Analysis revealed a significantly different change in fractional anisotropy (FA) between the two groups during the follow-up interval. An increase in FA during the post injury time interval was detected in the AFG group, indicating a beneficial effect of AFG in the rehabilitation of injured axons. Using tractography, AFG was suggested to be helpful in the restoration of fibers in SCI lesions, thus leading to promoted functional recovery.