Cardiovascular disease (CVD) is identified as the leading cause of death because of heart disease, stroke and other chronic diseases. The early and rapid detection of Troponin I, a cardiovascular disease biomarker, has an imperative role to prevent high risk of death. A lateral flow immunoassay (LFA) for detecting Troponin I quickly and effectively would provide yes/no answers with visual assessment, but not adequate to monitor the early stage of CVD. The Surface-Enhanced Raman Scattering (SERS) technique, offering highly sensitive and quantitative analysis, is integrated with LFA in order to measure Troponin I in a highly specific manner. Although the application of LFA is a modern technique, the development of SERS-based LFA is not straightforward. This research discusses three optimization procedures to develop SERS-based LFA for high sensitivity detection of Troponin I: (a) optimizing gold nanoparticle sizes (30, 50, 80, 100 nm) for SERS quantitative assay on Troponin I LFA; (b) investigating LFA components and fluid flow time to recognize Troponin I with SERS performance; and (c) evaluating different laser wavelengths and laser power for SERS-based LFA for the analysis of Troponin I. In a SERS-based LFA, these parameters are fundamental for augmenting sensitivity and detection limit. The SERS-based LFA became more sensitive than visual detection with naked eyes for quantitative quantification of Troponin I after optimization. In addition, the discussed procedures may offer advantages for the development of SERS-based LFA to detect various biomarkers in a highly sensitive manner.