We report on Surface-enhanced Raman Spectroscopy (SERS) based detection of p-nitrophenol (PNP), the end product of an enzymatic reaction of the acetylcholinesterase and the paraoxon (an organophosphorus insecticide), in a ppt/ppm concentration range using surface-immobilized Ag nanoparticles. Ag nanoparticles were synthesized by a modified Lee and Meisel method, and immobilized on the glass cover slips using the intermediate layer of an adsorbed polycation. Our XPS results indicate that significant oxidation of the Ag nanoparticle surface occurred when the substrate was kept in water under ambient conditions, and that such oxidation could be inhibited by purging the aqueous solutions with argon gas. We found that the oxidation of the Ag nanoparticle surface strongly affected the adsorption and orientation of PNP molecules. Specifically, vibrational spectra of argon-purged substrates indicated flat orientation of PNP molecules at sparse surface coverages, which was not observed with oxidized substrates. The oxidation state of Ag nanoparticles also had a dramatic effect on the limit of detection (LOD) of PNP, with the LOD of ~1 ppt and ~10 ppb for the argon-purged and the oxidized Ag surfaces, respectively. Significantly lower detection limit and the flat orientation of PNP molecules at unoxidized Ag surface both reflect stronger binding of PNP with the pristine Ag surface, probably due to charge transfer interactions between the aromatic ring of PNP molecules and the metal surface.