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Lateral flow immunoassay (LFIA) is a rapid and simple point-of-care method for the detection of various analytes. In the colorimetric sandwich format, the reaction outcome is a colored test zone line formed by Au nanoparticle (AuNP) conjugates captured by bound analyte molecules. Although nanoparticle design is crucial for the assay sensitivity, the correlation between the test zone brightness and the number and size of captured AuNPs has not been studied in detail. To fill this gap, we used an unprecedented set of 10 spherical and monodisperse AuNPs with diameters d ranging from 16 to 115 nm. The calculated optical properties are in excellent agreement with two-layered Mie theory for Au cores coated with 3 nm CTAC shells with a refractive index of 1.5. Different concentrations of AuNPs (ICP-MS and UV–vis measurements) were spotted onto a nitrocellulose LFIA membrane, and the color intensity of the spots was measured and analyzed with RGB and HSV color parameters. The minimal detected spot intensity was proportional to the surface nanoparticle density and the particle volume. The derived size dependence means that extinction rather than scattering is the main physical mechanism behind spot brightness. The limit of detection (LOD) in terms of the surface nanoparticle density scaled like the inverse third power of the particle size (more precisely, like ∼d–3.1) and was about 7 × 107 and 1.5 × 105 particles/mm2 for 16 and 115 nm AuNPs, respectively. We analyzed an ideal LFIA format, when one analyte molecule delivers just one AuNP to the test zone. In this case, the theoretical LODs were in the pg/mL range for a typical LFIA format with 0.1 mL of a 25 kDa analyte. In practice, these estimations could be increased by 2 orders of magnitude because of the larger ratio of analyte to captured AuNPs. This strongly reduced the assay sensitivity to the the ng/mL level. Although an ideal LFIA predicts a strong increase in the assay sensitivity with the AuNP size (scales like ∼d3.1), this improvement could be compensated for in part by an increase in the number of ineffective analyte molecules bound to the AuNP surface (scales like ∼d2).