In the present study, a novel tunable two-dimensional thermo-plasmonic grating based on gold nanorods was demonstrated by combining the plasmonic properties of the gold nanostructure and the applied external voltage. In this structure, a thin layer of the gold grating was typically deposited on a patterned polydimethylsiloxane substrate using the nanoimprint lithography method. The surface plasmon resonance of the fabricated plasmonic structure was excited by the surface plasmon imaging system based on a high numerical aperture objective lens and the charged coupled device camera. Based on the results, the number of the plasmonic hot spots due to the thermo-plasmonic effect increased by the external voltage, leading to an increase in this effect. Therefore, this reversible and tunable temperature confinement can be used as the controller of each element including cells in a defined micro-position.