Currently, viruses are utilized as vectors for gene therapy, since they transport across cellular membranes, escape endosomes, and effectively deliver genes to the nucleus. The disadvantage of using viruses for gene therapy is their immune response. Therefore, nanospheres have been formulated as a nonviral gene vector by blending l-tyrosine-polyphosphate (LTP) with polyethylene glycol grafted to chitosan (PEG-g-CHN) and linear polyethylenimine (LPEI) conjugated to plasmid DNA (pDNA). PEG-g-CHN stabilizes the emulsion and prevents nanosphere coalescence. LPEI protects pDNA degradation during nanosphere formation, provides endosomal escape, and enhances gene expression. Previous studies show that LTP degrades within seven days and is appropriate for intracellular gene delivery. These nanospheres prepared by water-oil emulsion by sonication and solvent evaporation show diameters between 100 and 600 nm. Also, dynamic laser light scattering shows that nanospheres completely degrade after seven days. The sustained release of pDNA and pDNA-LPEI polyplexes is confirmed through electrophoresis and PicoGreen assay. A LIVE/DEAD cell viability assay shows that nanosphere viability is comparable to that of buffers. X-Gal staining shows a sustained transfection for 11 days using human fibroblasts. This result is sustained longer than pDNA-LPEI and pDNA-FuGENE 6 complexes. Therefore, LTP-pDNA nanospheres exhibit controlled transfection and can be used as a nonviral gene delivery vector.