<p>Compared to mechanical rheology, micro-rheology (µR) can probe the viscoelasticity of soft matter non-invasively with spatial resolution and broad temporal coverage; however, the measurement quality is undermined by interference from the structural and dynamic inhomogeneity of the tested media. Herein, gold nanorods are dispersed in tested media as tracer particles, and their diffusive dynamics are monitored by depolarized dynamic light scattering for the analysis of the rheological properties of the tested media, as the rotational/translational dynamics of tracers can be converted to shear modulus <i>via</i> the generalized Stokes-Einstein relation. Because of their strong optical scattering to the laser and the polarization of incident light, the contrast in the dynamics of gold nanorods over the media can be enhanced, rendering the fast and accurate measurement of rheological properties. The method was verified for applications in broad types of substrates, including ergodic systems such as polymer solutions, silica suspensions, non-ergodic gel systems, and biological fluids such as plasma. The critical experimental parameters, for example, tracer size and scattering angle range, are studied for their impact on the measurement quality, and they can be systematically optimized for feasible and practical applications of the developed µR method.</p>