Objective To investigate the application of cervical diffusion tensor imaging (DTI) in the diagnosis of amyotrophic lateral sclerosis (ALS). Methods Cervical DTI and conventional magnetic resonance imaging (MRI) was performed in 28 ALS patients rated with ALS functional rating scale (ALSFRS) and 20 healthy control subjects to obtain the mean diffusivity (MD) and fractional anisotropy (FA) histograms of the cervical spinal cord. Correlation analysis between DTI of the cervical spinal cord and the ALSFRS scores of the ALS patients was conducted. Results Compared with the control subjects, the ALS patients had significantly lowered mean FA and reduced cross-sectional area of the cervical spinal cord (P<0.05), but the MD value did not differ significantly between the two groups (P>0.05). A close correlation was found between the mean FA of the cervical spinal cord and the ALSFRS score (r=0.730,P=0.000), but not between the MD value and the ALSFRS score in the ALS patients. Conclusion Patients with ALS have significantly lowered mean FA, which may serve as an indicator for the diagnosis orALS. Together with the ALSFRS score, cervical DTI can better characterize the progression of ALS.