We report a one-pot synthesis of hyperbranched polyglycerols possessing amino functionality by using a novel Boc-protected aminoethanol glycidyl ether monomer (BAG). A series of hyperbranched Boc-protected polyamino glycerols (PBAG) were prepared through a one-pot anionic ring opening multibranching polymerization to yield PBAG with controlled molecular weights (3500–17400 g/mol) and relatively low molecular weight distributions (1.2–1.8). Subsequent deprotection of PBAG yielded hyperbranched polyamino glycerols (PAG) with a globular polymeric structure that comprises a randomly branched structure with a large number of amine and hydroxyl groups. 1H, 13C, and 15N NMR, GPC and MALDI-TOF measurements confirmed the successful polymerization of the hyperbranched PAG polymers. With its superior biocompatibility of PAG, we anticipate the prospective potentials for the applications in biological and biomedical fields.