Miscanthus lutarioriparius, a domestic species of China, belongs to the genus of perennial rhizomatous Miscanthus and is a promising energy crop for biomass production. To establish an Agrobacterium tumefaciens transformation protocol and obtain transgenic plants co-expressing the Cry 2Aa^# and Bar genes, an Agrobacterium-mediated transformation method for M. lutarioriparius was developed in this study. To inhibit tissue browning, optimized callus induction and regeneration media were developed. Embryogenic calli were efficiently induced from immature inflorescences on Murashige & Skoog (MS) medium containing 0.2 g L^-1 citric acid. Agrobacterium tumefaciens EHA105 carrying pC3300 with the Cry 2Aa^# and Bar genes were introduced into embryogenic calli. Six transgenic lines were selected by Basta screening and confirmed by genomic PCR. The herbicide tolerance of M. lutarioriparius transgenic plants was verified by spraying with glufosinate and expression of the Cry 2Aa^# protein by ELISA. Therefore, an Agrobacterium-mediated transformation protocol for M. lutarioriparius was established and the co-expression of herbicide tolerance and Cry 2Aa^# was successfully introduced with the system.