Employing single nanoparticle detection with a laboratory-built high-sensitivity flow cytometer, we developed a simple and versatile platform that is capable of detecting the surface plasmon resonance scattering of gold nanoparticles (GNPs) as small as 24 nm, differentiating GNPs of different sizes, and providing accurate quantification of GNPs. Low-concentration samples (fM to pM) in small volumes (microL) can be measured in minutes with an analysis rate of up to 100-200 GNPs per second. Among these features, absolute quantification provides a distinct advantage because it does not require standard samples.