Optical sensors based on Surface-enhanced Raman scattering (SERS) effect are among the most versatile sensors due to their ability to characterize samples in various states of matter. The appeal of the SERS sensors lies in the molecular “fingerprint” specificity, sensitivity, and the non-invasive nature of the analysis. Although the current state of art SERS sensors have advanced toward ultrasensitivity with single-molecule detection limit, ultrafast analysis at femtosecond and sub-nanometer resolution, the application of these innovations in the industrial settings is still limited by the complexity of the substrate fabrication and the reproducibility of the SERS measurements. In this context, a study on the SERS sensors fabrication strategies and reliability of the SERS analysis is essential. This dissertation investigates various hybrids of noble metal and semiconductor materials and surface modifications to improve the stability and reliability of SERS measurement. Different industrial applications, including detection of petrochemical organic compounds and sensitive biochemical samples, were conducted to evaluate the performance of different SERS sensor designs. Evaluation of the morphology and surface functionalization of the substrate was accomplished to optimize the performance and stability of the collected signal. Together with the separately performed studies on Raman signal processing and interpretation, the proposed SERS sensor fabrication and signal analysis approach was successfully applied to detect and quantify organic isomers compounds and mutation point in peptides. The findings presented in this thesis offer rational SERS substrate designs and detection approaches that can advance the future commercialization of SERS sensors.