Nanomedicines are engineered nanoscale structures that have an extensive range of application in the diagnosis and therapy of many diseases. Despite the rapid progress in and tremendous potential of nanomedicines, their clinical translational process is still slow, owing to the difficulty in understanding, evaluating, and predicting their behavior in complex living organisms. Microfluidic techniques offer a promising way to resolve these challenges. Carefully designed microfluidic chips enable in vivo microenvironment simulation and high-throughput analysis, thus providing robust platforms for nanomedicine evaluation. Here, we summarize the recent developments and achievements in microfluidic methods for nanomedicine evaluation, categorized into four sections based on their target systems: single cell, multicellular system, organ, and organism levels. Finally, we provide our perspectives on the challenges and future directions of microfluidics-based nanomedicine evaluation.