A novel optical biosensor with gold surface bound silver nanoparticles has been constructed for detection of oncogene of c-Myc tagged DNA plasmid, which exhibited distinct optical properties of localized surface plasmon resonance. The silver colloidal nanoparticles were prepared by using chemical reduction method with sodium citrate, starch and silver nitrate, which were characterized by using UV-Vis absorption spectroscopy, transmission electron microscopy, and cyclic voltammetry, indicating that the silver colloidals were pure, sphere-shaped and narrow- dispersed size distribution with a diameter of 11.70 ± 1.94 nm. The silver nanoparticles were well assembled on the gold substrate with 1,4-dithiothreitol. In the subsequent linking step with compounds of L-cysteine, 3-mercaptopropionic acid, and sodium thioglycolate/HCl, L-cysteine exhibited best characteristics for linking with Ag nanoparticles and formed a nice sensing film by conjugating with a antibody of c-Myc (3C7), mouse monoclonal IgG. The c-Myc antibody immobilized optical biosensor can be well applied to determination of c-Myc tagged DNA plasmid with good regeneration ability. The detection limit was evaluated to be 1.0 pg/μL.