The invention discloses a graphene film sensitized D-shaped optical fiber surface plasmon resonance (SPR) sensor. The sensor comprises a D-shaped optical fiber, wherein a silver film layer is arranged on the polished surface of the D-shaped optical fiber; and a graphene film layer is arranged on the surface of the silver film layer. A preparation method comprises the following steps of: (1) preparing the D-shaped optical fiber, and preparing the silver film layer on the polished surface of the D-shaped optical fiber; and (2) preparing the graphene film layer on the surface of the silver film layer. The D-shaped optical fiber is used as a light transmission medium, an SPR structure is formed by plating a silver film on the surface of the D-shaped optical fiber, the sensitivity of the SPR sensing structure is increased by depositing or growing a graphene film material on the surface of the silver film, and the sensor has the prominent advantages of small volume, light weight, high sensitivity, quick response and the like, and has wide application prospects in the aspect of detecting trace gas, liquid, chemical elements, deoxyribonucleic acid (DNA) and the like in the fields of biology, chemistry, medicine and the like.