Chemokine receptor-4 (CXCR4) has been demonstrated to be overexpressed in glioma cells and associated with poor survival. In the present study, we designed and synthesized 99mTc-labeled low-generation dendrimer-entrapped gold nanoparticles (Au DENPs), which were modified with CXCR4-ligand for targeted single photon emission computed tomography (SPECT)/computed tomography (CT) imaging of glioma. Generation two poly(amidoamine) dendrimers were sequentially conjugated with diethylenetriamine pentacetate acid (DTPA) and polyethylene glycol (PEG) modified with FC131 peptide (a CXCR4-specific ligand), and were then utilized as a template for entrapment of gold nanoparticles (Au NPs). After radiolabeling with 99mTc via DTPA chelation, the glioma-targeting SPECT/CT dual mode nanoprobe {(Au0)6-G2-99m-DTPA-(PEG-FC131)} DENPs were generated. The FC131-functionalized Au DENPs before and after 99mTc radiolabeling were well characterized. The results revealed that prior to 99mTc radiolabeling, FC131-functionalized Au DENPs had a uniform size distribution, acceptable biocompatibility in the given Au concentration range and could specifically target glioma cells due to the attached FC131. Furthermore, the FC131-functionalized Au DENPs could be readily radiolabeled by 99m Tc with good radiochemical stability and high radiochemical purity. The {(Au0)6-G2-99mTc-DTPA-(PEG-FC131)} DENPs appeared to be effective in targeted SPECT/CT imaging of glioma cells in vitro and in vivo in a subcutaneous tumor model. The developed FC131-functionalized dendrimer-based nanoprobe may hold a great potential for utilization in SPECT/CT imaging of different CXCR4-overexpressing cancers.