Guanidine-rich macromolecules show diverse bioactivities, and guanidine group-rich dendritic macromolecules often exhibit superior biological properties compared to linear ones with the same or similar number of guanidine groups. However, guanidine-rich dendritic macromolecules were usually synthesized through tedious and expensive procedures, in which toxic organic solvents had to be used. In the present work, we developed a twostep method to synthesize hyperbranched polymers with periphery guanidine groups without using any organic solvents. Hyperbranched polylysines were first synthesized by thermal polymerization of L-lysine hydrochloride in the presence of KOH. The molecular weight of the hyperbranched polylysines increased with polymerization time. Guanidine-rich hyperbranched polymers were then prepared by treating the hyperbranched polylysines with 1H-pyrazole-1-carboxamidine hydrochloride in the presence of a base using water as the solvent, in which the free amino groups of the hyperbranched polylysines were transformed into guanidine groups. The guanidinerich hyperbranched polymers showed lower cytotoxicity and hemolytic activity than the corresponding precursor hyperbranched polylysines. Co-incubation of the guanidine-rich hyperbranched polymers and doxorubicin with pancreatic cancer cells (PANC-1) showed enhanced cellular uptake of the drug. The polymers also showed the ability to promote penetration of doxorubicin deeply into multicellular PANC-1 spheroids upon coincubation. Co-administration of the guanidine-rich hyperbranched polymers with doxorubicin and gemcitabine, respectively, greatly enhanced the anticancer activity of the drugs in PANC-1 xenograft mouse models.