The combination of a surface acoustic wave (SAW) biosensor with graphene oxide (GO) provides a promising perspective for detecting DNA mutation. The GO-modified SAW biosensor was prepared by conjugating GO onto the SAW chip surface via electrostatic interaction. Afterward, the probe was immobilized on the GO surface, and detection of DNA mutation was realized by hybridization. The hybridization with a variety of targets would yield different mass and conformational changes on the chip surface, causing the different SAW signals in real time. A total of 137 clinical samples were detected by a single-shot analytical assay based on GO-modified SAW biosensor and direct sequencing in parallel. The diagnostic performance (both sensitivity and specificity) of the assay was evaluated with the direct sequencing as a reference testing method. The phase-shift value of three genotypes in 137 clinical samples was significantly different (p < 0.001). Furthermore, testing of diagnostic performance yielded diagnostic sensitivity and specificity of 100% and 88.6% for identifying CT and CC genotype, 98.0% and 96.2% for identifying CT and TT genotype, respectively. The single-shot analytical assay based on the GO-modified SAW biosensor could be exploited as a potential useful tool to identify CYP2D6*10 polymorphisms in clinical practice of personalized medicine.