Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.