We describe a facile approach to synthesizing acetylated dendrimer-entrapped gold nanoparticles (Au DENPs) with enhanced Au loading in the dendrimer interior. In this study, amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers (G5.NH2) were used as templates to form Au DENPs via a stepwise Au salt complexation/reduction approach, followed by acetylation of the dendrimer terminal amines. The formed Au DENPs before and after acetylation were characterized with different techniques. We show that the stepwise complexation/reduction of HAuCl4 is able to significantly improve the loading amount of Au within the dendrimer interior. UV-Vis spectroscopy reveals that the intensity of the surface plasmon resonance (SPR) band increases with the Au loading, confirming the stepwise loading synthesis of Au DENPs. TEM images show that the synthesized Au DENPs have a quite uniform size distribution with sizes tunable in the range of 2–4 nm depending on the Au loading. The formed acetylated Au DENPs with enhanced Au loading are very stable under different pH and temperature conditions. Importantly, computed tomography (CT) imaging experiments reveal that the formed acetylated Au DENPs have higher attenuation intensity than a clinically used iodinated contrast agent, Omnipaque, at the same molar concentration of active elements (Au or iodine), and enable significantly enhanced CT imaging of rat heart in vivo. The acetylated Au DENPs with enhanced Au loading formed via the facile stepwise approach may be used as contrast agents for highly sensitive CT imaging applications.