Gold nanorods have strong surface plasmon band in the near-infrared region, at which light penetrates deeply into tissues, and convert the absorbed light energy into heat. Therefore, gold nanorods are expected to act as an effective contrast agent for in vivo bioimaging and as a photosensitizer for photothermal therapy. In order to efficiently achieve these applications without side effects, gold nanorods should be selectively accumulated in the target organ. In this study, we optimized the length of the poly (ethylene glycol) (PEG) chain to stabilize the PEG-modified gold nanorods in blood circulation, and investigated the effects of PEG grafting level and injection dose on the biodistribution and enhanced permeability and retention (EPR) effect after intravenous injection into mice. PEG5,000-, PEG10,000- and PEG20,000-modified gold nanorods showed higher blood circulation stability compared with PEG2,000-modifed gold nanorods. Higher PEG grafting levels were advantageous for the reticuloendothelial system (RES) avoidance and suppression of aggregation of the gold nanorods in the blood circulation. Modification with a PEG5,000: gold molar ratio of 1.5 was sufficient to show both prolonged circulation and the EPR effect. When the injection dose was increased above 39.0 μg of gold, the RES uptake in the liver was saturated and surplus gold nanorods were distributed to other tissues, especially the spleen and tumor. This information is important key to provide the successful application of gold nanorods in the field of nanomedicine.