For biomedical application of nanoparticles, the surface chemical functionality is very important to impart additional functions, such as solubility and stability in a physiological environment, and targeting specificity as an imaging probe and a drug carrier. Although polyethylene glycol (PEG) has been used extensively, here, it is proposed that hyperbranched polyglycerol (PG) is a good or even better alternative to PEG. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared using a polyol method are directly functionalized with PG through ring-opening polymerization of glycidol. The resulting SPION-PG is highly soluble in pure water (>40 mg mL−1) and in a phosphate buffer solution (>25 mg mL−1). Such high solubility enables separation of SPION-PG according to size using size exclusion chromatography (SEC). The size-separated SPION-PG shows a gradual increase in transverse relaxivity (r2) with increasing particle size. For biological application, SPION-PG is functionalized through multistep organic transformations (–OH → –OTs (tosylate) → –N3 → –RGD) including click chemistry as a key step to impart targeting specificity by immobilization of cyclic RGD peptide (Arg-Gly-Asp-D-Tyr-Lys) on the surface. The targeting effect is demonstrated by the cell experiments; SPION-PG-RGD is taken up by the cells overexpressing αvβ3-integrin such as U87MG and A549.