Abstract: Fluorescence resonance energy transfer (FRET) effect of graphene oxide on DNA fluorescent molecular probe is a very important factor of DNA the biosensor, which is related to the detection sensitivity of graphene oxide based DNA biosensors. The effects of the concentration of graphene oxide, the reaction temperature, and the length of DNA on FRET were investigated. The results show that the concentration of graphene oxide, the reaction temperature, and the length of DNA play significant roles on FRET effects. The fluorescence quenching efficiency of DNA molecule probe was improved with the increasing of graphene oxide concentration and the reaction temperature, with the decreasing of the length of DNA. Therefore, in order to get the excellent biosensor, the concentration of graphene oxide, the reaction temperature, and the length of DNA should be taken into account in the design of graphene oxide DNA biosensor.