The invention discloses a DNA detection method based on a target-DNA-triggered layer-by-layer self-assembly graphene-based photoelectric biosensor and application. A layer-by-layer self-assembly DNA /graphene composite is formed by use of DNA intermolecular interactions for triggering of graphene self-assembly, the agglomeration of the graphene can be effectively overcome under the condition of no introduction of other metal nano materials, the prepared layer-by-layer self-assembly DNA /graphene composite exhibits excellent chemical catalytic activity and chemical stability, and is conducive to the subsequent electrochemical and photochemical detection of target DNA; and the biosensor has the advantages of simple preparation, low price, good stability, high sensitivity, wide linear range, good selectivity and the like.