Gene therapy is a good alternative to drugs in chemotherapy due to drug resistance and toxicity and known to lower the probability of mutation of cells through gene carrier. Herein, gene carrier nanoparticles with minimal toxicity and high transfection efficiency were fabricated from biodegradable polymer (L-tyrosine polyurethane, LTU) which was presynthesized from desaminotyrosyl tyrosine hexyl ester (DTH), and polyethylene glycol (PEG) through double emulsion method and used to evaluate their potential biological activities molecualr controlled release and transfection studies. In order to evaluate cell adsorption and transfection of nanoparticles, two types of nanoparticles were prepared, the one from fluoresced using fluorescently labeled bovine serum albumin (FITC-BSA) to investigate cell adsorption, and the other from encapsulated with DNA-linear polyethylenimine (LPEI) complex to investigate the transfection efficiency in LX2 (human hepatic stellate cell), HepG2 (human liver cancer cell), MCF7 (human breast cancer cell). The morphology of these nanoparticles, which was confirmed both by microscope image and TEM, was a spherical shape with an average diameter of about 260 nm and 145 nm. The biodegradable nanoparticles showed the typical features of double emulsion. After 14 days, DNA in nanoparticles have been released from the LTU nanoparticles. Furthermore, a successful cellular uptake of LTU nanoparticles in hepatic stellate cells has been confirmed and high transfection efficiency also confirmed in LX2, HepG2 cells with check proper concentration of LPEI due to toxicity. These characteristics are ideal for gene therapy designed to transport and release a drug into the cytoplasm.