The invention discloses a method for functional prediction of single nucleotide genomic variation in a non-coding region, which comprises the following steps: 1) identifying a chromatin open region; 2) identifying transcription factor bin sites; 3) assessing the role of single nucleotide variations: Based on the site-specific frequency matrix of transcription factors, the effect of single nucleotide variations in the transcription factor binding site region on transcription factor binding is calculated, and the single nucleotide variations that significantly change the binding capacity of transcription factors are identified; the role of single nucleotide variations is further evaluated by viewing the biological pathway of the target gene of the transcription factor. The method recognizesa variety of transcription factors and their binding sites at one time through chromatin open region information and gene expression information, and realizes the functional annotation of non-coding region genomic mutation.