Single-particle tracking (SPT) microscopy is increasingly used to characterize nanoparticulate systems. We introduce a concept for estimation of particle number concentration in Brownian particle dispersions using SPT based on a model for the trajectory length distribution of particles to estimate the detection region volume. The resulting method is independent of precalibration reference measurements, and robust with respect to image processing settings. Experimentally estimated concentrations of different dilutions of 0.19- and 0.52-μm polymer nanospheres are in excellent agreement with estimates computed from the concentrations of the stock solutions.