The non-specific interaction between the surface of gold nanorods (GNRs) and biomolecules is a significant problem in hyperthermia applications. In this work, a novel surface modification strategy was performed using zwitterionic oxidized glutathione (GSSG) to modify the GNRs through robust Au-S bond formation. The GSSG-GNRs were stable in aqueous solution over a broad range of pH and high ionic strength values. Importantly, the glutathione coating greatly reduced non-specific protein adsorption on GNRs due to zwitterions. In order to target tumor sites, folic acid (FA) and Rhodamine B (RBITC) dye were conjugated to GSSG modified GNRs through the reaction between the activated carboxyl group and the amino group. The targeting of RBITC-FA-GSSG-GNRs in human cervical carcinoma cells (HeLa cells) with high-level folate receptor expression was confirmed using confocal laser scanning microscopy (CLSM) to excite Rhodamine B dye at 488 nm. The targeting photothermal therapy efficacy was monitored in real time with the multi-channel function of a confocal laser scanning microscope coupled with an 808 nm laser. The results indicate that the FA-GSSG-GNR hybrids demonstrate efficient photothermal conversion and excellent targeting hyperthermia, and are therefore suitable for biomedical applications.