A family of water soluble and positively charged L-arginine based poly(ester amide)s (Arg-PEAs) was synthesized and characterized. These biodegradable polymers consist of three nontoxic building blocks: L-arginine, diols, and dicarboxylic acids. The Arg-PEAs were prepared by solution polycondensation reaction of tetra-p-toluenesulfonic acids salts of bis-(L-arginine) α, ω-alkylene diesters and di-p-nitrophenyl esters of dicarboxylic acids. Optimal conditions of the monomers and polymers synthesis were investigated, and the monomers and Arg-PEAs were chemically characterized. Arg-PEAs were found to have good solubility in water and many other polar solvents. Structure-function relationship of the Arg-PEAs revealed that changing the number of methylene groups in the diol or/and diacid segment could finely tune the hydrophobic and cationic properties of the Arg-PEAs. MTT assay showed that all the prepared Arg-PEAs were non-toxic to the cell lines even at very large doses. Arg-PEAs with double bond functionality could be photo-crosslinked with polyethylene glycol diacrylate to form cationic hybrid hydrogels.