In recent decades, advanced therapeutic modalities such as therapeutic cells, viral vectors, and extracellular vesicles (exosomes), have emerged as effective therapies for intractable diseases. These therapeutic modalities produced through bioprocesses must be purified from contaminants. Effective separation methods are essential for optimizing therapeutic modalities. This review highlights innovative temperature-modulated separation methods enabled by the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAAm). The design of PNIPAAm-modified interfaces plays a pivotal role in ensuring precise and efficient separation. We summarize the recent advancements in the application of temperature-modulated separation methods for cells, viral vectors, and exosomes, with a focus on the design of PNIPAAm interfaces.