Polyethylene glycol (PEG) is a multifunctional polymer that has many uses in medical and biological applications. Recently, PEG has been mainly used in developing nanomaterial-based drug delivery systems (DDS). PEG is characterized by its high solubility, biological inertness, and ability to escape from immune cells (stealthiness) after systemic injection. The most challenging problem for PEGylated nanomaterials is their rapid elimination from the bloodstream after repeated doses of systemic injection, called accelerated blood clearance (ABC). Therefore, in this study, the effect of PEGylated nanomaterial dose concentration on ABC induction will be investigated using quantitative, histological, and immunohistochemical analyses. A higher dose concentration (2 mg/kg) of PEGylated gold nanoparticles (PEG-coated AuNPs) reduced the ABC phenomenon when intravenously injected into mice preinjected with the same dose. In contrast, a lower dose concentration (< 1 mg/kg) significantly induced the ABC phenomenon by the rapid elimination of the second dose of PEG-coated AuNPs from the bloodstream. To explain the relationship between the dose concentration (from PEG and AuNPs) and the induction of ABC, the biodistribution of PEG-coated AuNPs in liver and spleen [reticuloendothelial systems (RES)-rich organs] was investigated. The injected dose of PEG-coated AuNPs accumulated mainly in the hepatic Kupffer cells and hepatocytes. Similarly, spleen red pulp received a higher amount of the injected dose of PEG-coated AuNPs. However, the biodistriution profiles of PEG-coated AuNPs after the first and second dose for different dose concentrations varied in RES-rich organs. Additionally, the number of B lymphocytes, which have an important role in producing anti-PEG immunoglobulin (Ig)M, was affected by the repeated dose of PEG-coated AuNPs in the spleen. Therefore, for effective nanomaterial-based DDS development, dose optimization of PEG molecules that express PEGylated nanomaterials is important to reduce the ABC phenomenon effect. The ideal concentration of PEG molecules used to coat nanomaterials and the role of RES-rich organs must be determined to control the ABC phenomenon effect of PEGylated nanomaterials.