The surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip has been considered a high-sensitivity sensor that can overcome the low sensitivity and the difficulty of quantitative analysis problems inherent in the colorimetric LFA sensor. In the SERS-based LFA strip reported so far, a liquid sample flows through the nitrocellulose membrane in a single pathway. In some cases, however, this single-flow approach still has a limitation in detection sensitivity. This study developed a conceptually new SERS-based dual-flow LFA sensor to improve the detection sensitivity in a single-flow LFA sensor. First, a 25 nm Raman reporter-labeled gold nanoparticle (AuNP) solution flowed through one way, and a 45 nm AuNP solution continuously flowed through another path. This sequential flow of two different AuNP solutions enables forming additional bright hot spots between 25 and 45 nm AuNPs in the test line, and the SERS signal is strongly enhanced. Using this SERS-based dual-flow LFA sensor, it was possible to detect thyroid-stimulating hormone less than 0.5 μIU/mL that cannot be measured with a SERS-based single-flow LFA sensor.