Abstract: A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN19, pGFP, and pCHS, which included gfp gene driven by the CaMV 35S promoter. The hairy roots of Petunia hybrida were induced by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 45%. The PCR results showed that rolB from K599 Ri plasmid and gfp from pBIN-35S-GFP were co-transformed into the genome of P. hybrida. The high activity of green fluo-rescence protein was detected by fluorescence microscopy. In particularly, the vector carries multiple cloning sites at both 5′ and 3′ of the CaMV 35S promoter, which allows easy exchange 35S promoter to study other promoter functions. In addition, there are multiple cloning sites at 5′ end and one-sites of EcoRⅠand BsmⅠsites at 3′ end of gfp. Therefore, it supports to fusion target genes to expression fusion protein and can be replaced with any other genes of interest for genetic transforma-tion.