The pH-responsive amphiphilic polyphosphazenes bearing N,N-diisopropylethylenediamine (DPA) have been proven to be promising nanovehicles for drug antitumor therapy. To further modify these amphiphilic polyphosphazenes with fluorescent labeling agent or other biochemical functional groups, serine methyl ester containing active chemical group NH(2) was chosen to be introduced to get a novel polymer [NP(PEG)(0.24) (DPA)(0.5)(SME)(1.26) (n) (PDS-NH(2) ). Considering the possible toxic effect of -NH(2) group, the biocompatibility in bloodstream and nanotoxicity on human normal hepatic L-02 cells was evaluated in this study. The polymer NP(PEG)(0.24)(DPA)(0.5)(SME-BOC)(1.26) (PDS-BOC) linked with tert-butyloxycarbonyl groups to protect and hide -NH(2) group was applied as the comparison. First, the bovine serum albumin (BSA) adsorption and phagocytic uptake behavior in human THP-1 macrophages were performed. The results suggested that only a minor percentage of the nanoparticles were involved in BSA binding and phagocytic uptake as the result of PEGylation on the particulate surface. To determine the nanotoxicity on human normal hepatic L-02 cells, we measured cell viability, apoptosis and necrosis, reactive oxygen species generation, the loss of mitochondrial membrane potential, and the levels of the apoptotic signaling proteins in L-02 cells after the cells being exposed to nanoparticles of different concentrations (0.1, 0.2, and 0.5 mg/mL) for 24 h. Our data indicated that the two nanoparticles induced cytotoxicity in a dose-dependent manner; PDS-NH(2) caused more cytotoxicity than PDS-BOC as a result of -NH(2) exposure. The increased expression of caspase-3 and caspase-9 suggested that they triggered apoptosis through mitochondria-dependent pathways in L-02 cells.