Previous approaches of determining the molar concentration of nanoparticles often relied on the calibration factors extracted from standard samples or required prior knowledge regarding the geometry, optical, or chemical properties. In the present work, we proposed an absolute quantification method that determined the molar concentration of nano-objects without any calibration factor or prior knowledge. It was realized by monitoring the dynamic adsorption processes of individual nanoparticles with a high-speed surface plasmon resonance microscopy. In this case, diffusing nano-objects stochastically collided onto an adsorption interface and stayed there ("hit-n-stay" scenario), resulting in a semi-infinite diffusion system. The dynamic processes were analyzed with a theoretical model consisting of Fick's laws of diffusion and random-walk assumption. The quantification of molar concentration was achieved on the basis of an analytical expression, which involved only physical constants and experimental parameters. By using spherical polystyrene nanoparticles as a model, the present approach provided a molar concentration with excellent accuracy.