Temperature sensing is a promising method of enhancing the detection sensitivity of lateral flow immunoassay for point-of-care testing. A temperature increase of more than 100 °C can be readily achieved by photoexcitation of reporters like gold nanoparticles (GNPs) or colored latex beads (CLBs) on the strips with a laser power below 100 mW. Despite its promise, processes involved in the photothermal detection have not yet been well-characterized. Here, we provide a fundamental understanding of this thermometric assay by combining experiments and simulations using non-fluorescent CLBs as the reporters deposited on nitrocellulose membrane. By measuring the dependence of temperature rises on the number density of membrane-bound CLBs, we determined a 1.5-fold enhancement of the light absorption at 520 nm by the beads (diameter of 0.4 μm). The enhancement, however, was compromised by a 5-fold reduction of the incident laser power due to multiple scattering of the light in this highly porous medium. The limit of detection was measured to be 1 × 105 particles/mm2. In line with previous studies using GNPs as the reporters, the CLB-based thermometric assay provides a 10× higher sensitivity than color visualization, as demonstrated with the immunoassay for nucleocapsid proteins of the SARS-CoV-2 virus.