Abstract: The diffusion of nanoparticles in physiological media is very important in the process of life evolution, information transmission, and drug delivery. Physiological media such as mucus, tissue fluid, and cytoplasm not only have complex porous properties, but also often exhibit microscale flows related to life activities. The interaction between flow and diffusion is extremely complex, and is affected by the porous properties of physiological media. In the experiment, the microfluidic technology is used to construct a microscale flow environment of polymer solution, the particle tracking technology is employed to measure the movement of nanoparticles, the movement characteristics of nanoparticles are then characterized based on statistical characteristics, and the effects of the microscale flow on nanoparticle diffusion are analyzed. The results show that the microscale flow has an effect on the diffusion of nanoparticles in the direction of the flow and the direction of vertical flow; the restricted degree of nanoparticle diffusion is weakened in the flow direction, showing the multi-stage characteristics of sub-diffusion, Brownian diffusion to super-diffusion; the diffusion of nanoparticles shows an approximate Brownian characteristic in the direction of vertical flow, but the diffusion coefficient is significantly higher than that of the static case. The analysis reveals that the effect of microscale flow on the nanoparticles diffusion in polymer solution is mainly due to the change of the polymer network structure and dynamics. The research results can provide a certain reference for the interpretation of the transport mechanism of nanoparticles in physiological media, and the design and transport enhancement of nano-drugs.