Uranium is one of the important nuclear materials to nuclear industry. Because of the direct disposal of spent fuel, there is still a huge possibility that uranium migrates into the groundwater, causing water contamination. It is of great importance to understand the concentration and their species distribution in aqueous solutions. Surface-Enhanced Raman Scattering (SERS) technique has been widely used for the detection of uranium (Ⅵ). However, the interactions between uranium (Ⅵ) and SERS substrate cause the symmetric stretching vibration peak of uranium (Ⅵ) shift to low wave number direction, which is unfavorable for confirming the species of uranium (Ⅵ) in aqueous solution. For instance, the normal Raman bands of uranyl in nitric acid solution are 871 cm-1, which belongs to the symmetric stretching mode of UO2+2. However, it moves to 710 cm-1 on the surface of silver nanorods SERS substrtate. What’s more, different SERS substrate causes different number of shift. Graphene has advantages of inertness and integrity as well as 2-dimensional thickness. In this paper, graphene-isolated SERS substrate which is silver nanoparticles (AgNPs)/graphene complex substrate, was designed to prevent the interaction between SERS substrate and it was analyzed by using the inert graphene layer. First of all, according to our previous work, AgNPs SERS substrate was fabricated on silicon wafer by using an ascorbic acid-actived self-assembly method. Then, AgNPs/graphene complex substrate was prepared by transfering monolayer graphene onto the self-assembly AgNPs substrate. The morphology of complex substrate was obtained by SEM. Some AgNPs link together closely to form nanochain structures. Nanochain structures were distributed evenly on the surface of silicon wafer. The 2-dimensional thickness of graphene did not affect the morphology of AgNPs. When using the complex substrate to detect uranyl nitrate (5×10－4 mol·L-1), the Raman peak that appeared around 771 cm-1 is considered to be the symmetric stretching mode of UO2+2, shifting back about 52 cm-1 to high wave number direction when compared with AgNPs substrate, which was about ~719 cm-1. The result indicates that graphene layer isolates the interaction between AgNPs substrate and uranyl in some degree.