Detection of bioactive metabolites produced by bacteria is important for identifying biomarkers for infectious diseases. In this study, a surface-enhanced Raman spectroscopy-based technique was developed for the detection of bioactive metabolite indole produced by Escherichia coli in biological media. The use of highly sensitive Au@Ag core-shell nanoparticles resulted in the detection of indole concentration as low as 0.0886 mM in standard solution. The supplementation of growth media with 5 mM of exogenous tryptophan resulted in the production of a maximum yield of indole of 3.139 mM by E. coli O157:H7 at 37 °C. The growth of bacterial cells was reduced from 47.73 × 108 to 1.033 × 106 CFU/mL when the cells were grown in 0 and 10 mM exogenous tryptophan, respectively. The amount of indole in the Luria-Bertani (LB) media had an inverse correlation with the growth of cells, which resulted in a 3-log reduction in the colony-forming unit when the indole concentration in the media was 20 times higher than normal. This work demonstrates that surface-enhanced Raman spectroscopy is an effective and highly sensitive method for rapid detection of bioactive metabolites in biological matrix.