Recent anticancer drugs are made to be larger to selectively pass the tumor vessel and to stay in the interstitium. Understanding of the drug movement in a single molecule is indispensable to optimize drug delivery. Here we report the tracking of single nano-particles with different sizes in tumor xenografts in vivo. The motions of the nano-crystals were clearly observed and random with one-way movement. The analysis of the trajectory shows that contribution of diffusion and interstitial fluid current to the delivery processes. The diffusion coefficient was inversely related to the size and position and velocity was related to the position of the particles. Our data are basic and important for understanding pharmacokinetics and convenient finding for constructing novel drug delivery system.