A nano-scale biosensor chip surface was fabricated using dinitro-phenylated keyhole limpet hemocyanin (DNP-KLH) protein conjugate as ligand supported by underlying 11-amino 1-undecanethiol hydrochloride(AUT) self-assembled monolayer (SAM) and bis sulfosuccinimidyl suberate (BS3) as crosslinker, Bioactive thin films were fabricated over gold chip via layer-by-layer self- assembly methods. Biomolecular interaction between substrate specific anti-TNT antibody and DNP-KLH conjugate surface was monitored through surface plasmon resonance based optical sensor. The quantitation of tri-nitro toluene(TNT) on this bioactive surface was done using the solution based competitive inhibition assay. The DNP-KLH surface biosensor has shown a detection limit of 0.14 ng/ml (140 ppt) for TNT molecule. The detection limit of surface plasmon resonance (SPR) biosensor was further enhanced by using goat anti mouse antibody to the 0.002 ng/ml for TNT analyte. This TNT specific biosensor holds the promise to be one of most sensitive sensor surface under indirect competitive assay format, A short injection (12 sec) of 10 mM glycine-HCl solution was found adequate for regeneration of DNP-KLH surface for repeated use.