The purpose of this study was to use a convenient synthetic strategy to prepare a new family of biodegradable amino acid-based poly(ester amide)s (PEAs) with pendant amine groups along the polymer back-bone, and investigate the applications of the new polymers in the biomedical area. Two amino acids, L-phenylalanine (Phe) and L-lysine (Lys), were used as the model amino acid compounds to illustrate the synthesis, characterization, and biological property of this new family of functional PEAs. These new PEAs were obtained by two-step reactions, the ring-opening reaction of epsilon-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Z-LysNCA) with L-phenylalanine hexane-1,6-diol diester p-toluenesulfonate (Phe-6), and subsequently solution polycondensation with di-p-nitrophenyl sebacoyl (NS). The benzyloxycarbonyl (Z) protective groups of the resulting polymer (PEA-Z-Lys) were completely removed to produce the new functional PEAs having free pendant amine groups (PEA-Lys-NH2). The level of the pendant amine groups on the PEA-Lys-NH2 could be tailored by adjusting the Phe-6 to Z-LysNCA feed ratio. Analyses of FTIR, H-1 NMR, C-13 NMR spectra, and DSC revealed the desired chemical structures and thermal property of PEA-Z-Lys as well as the final functional PEA-Lys-NH2. The free pendant amine groups were used to chemically conjugate a fluorescent dye to demonstrate the utility of this new family of functional PEA. An in vitro cell culture study of these functional PEAs showed that they supported the proliferation of bovine aortic endothelial cell slightly better than gelatin-coated glass coverslips. This new family of biodegradable functional PEA with free amine groups may have great potential applications for biomedical and pharmacological fields.