In this study, the tms2 gene, the cause of crown gall, was detected in two Rosa hybrida L. (“Pearl” and “Angelina”) cultivars using electrochemical methods. In this study, stabilization and hybridization of single-strand DNA (ssDNA) related to tms2 gene, Agrobacterium tumefaciens was investigated by electrochemical impedance spectroscopy. For this purpose, the pencil graphite electrode was modified with nanomaterials including reduced graphene oxide and gold nanoparticles. The nanobiosensor could be used to detect infected samples of “Angelina” and “Pearl” roses. DNA/DNA hybridization was performed by placing an electrode modified with ssDNA at a concentration of two (µmol/L) target DNA. The results showed proper stabilization and hybridization of DNA at the electrode surface. The designed biosensor has good selectivity. DNA-based nanosensor has advantages such as low cost, simplicity and scalability and can be the basis for the development of genomic detection tools.