N, N-dimethylcysteamine modified PATU (PATU-DMCA) was synthesized through thiol-ene Michael addition reaction based on a novel dendrimer of polyacylthiourea (PATU). NMR results show that the dendrimer structure of PATU-DMCA is correct and integrated. Agarose gel electrophoresis assay and dynamic light scattering results demonstrate that PATU-DMCA of low generations with low molar ratios of N and P can package DNA to form nanoparticles with size of 60~100 nm and Zeta potential of 10~20 mV. In human cervical carcinoma cell HeLa and human lung cancer cell A549, the formed polyplexes with low molar ratios of N and P show higher transfection efficiency in serum-free medium than the classical dendrimer poly (amidoamine), while the cytotoxicity is equal to each other. The subcellular distribution study in HeLa indicate that the intracellular trafficking of the polyplexes include cell membrane adhesion, endocytosis into cell, entering into lysosome, and lysosomal escape via ‘proton sponge effect’ for transfection in cytoplasm and nucleus. Both PATU-DMCA and PATU itself have enormous potential for gene delivery.