Endotoxins, the toxic substances derived from gram-negative bacterial cell lysis can be lethal even in the ng/Kg of body weight. The conventional method to detect LPS is Limulus amebocyte lysate (LAL) test, which uses an enzyme found from the horseshoe crab, thereby endangering the species. In present work, Surface Enhanced Raman Spectroscopy (SERS) is the proposed method that addresses this ecological issue and provides a simple, a label-free, cost-effective, reliable, rapid, selective, and sensitive method is used to detect endotoxin (biochemically known as lipopolysaccharides; LPS). Chemometric method is used to analyse a larger SERS dataset. In this work, a positively charged Ag/Au bimetallic nanoparticles are prepared using reduction method and performed SERS experiments with excellent reproducibility of spectra and detection capabilty up to the limit of pg/mL. In this work, SERS detection of LPS extracted from three bacteria; E. coli, Salmonella Typhimurium and Pseudomonas sp bacteria, is carried out that eluciates SERS as a potential technique. In course of study, we optimize sampling protocol for getting reproducible SERS signal and find out biochemical marker band for LPS. Next SERS experiments is also carried out to discriminate and detect LPS in the presence of other biomolecules. Multivariate analysis is used to obtain semi-quantitative analysis.