Novel polymers composed of net-like PEGylated poly(β-amino ester) (N-P-1, M(w) = 6900 or N-P-2, M(w) = 21,400) and oligoethylenimine (OEI) (OEI423 or OEI600) were synthesized and evaluated as gene carriers. The molecular weights of these polymers were well-controlled by the concentration of the cross-linking reaction. The synthesized polymers showed high biodegradability, less cytotoxicity, and efficient DNA retard ability. The N-P-1-OEI600/DNA complex showed much slower aggregation in the presence of 10 and 20% serum solutions. In vitro transfection assays, N-P-2-OEI423, N-P-1-OEI600, and N-P-2-OEI600 showed enhanced transfection efficiency compared with the PEI25K control in the presence or in the absence of serum in different cell lines. In particular, in Cos-7 cells, the transfection efficiency of N-P-1-OEI600 was 20.9 times higher than that of PEI25K in the presence of serum. The polymer/DNA complex stability, lower cytotoxicity, and higher transfection efficiency in the presence of serum revealed that N-P-1-OEI600 could be a potential nonviral gene carrier for In Vivo application.