Nanofiltration (NF) with advantages of high efficiency and low‐cost has attracted increasing attentions in bio‐separation. However, the large‐scale application is limited by the inferior molecular selectivity, low chemical stability and serious membrane fouling. Many efforts, thus, have been devoted in NF materials design for specific applications to enhance the separation efficiency of bio‐products and increase membrane life‐time, as well as reduce the operating cost. This review summarized the recent progress of NF applications in bio‐separation, discussed various demands for NF membrane in the bio‐products purification and corresponding material innovations, finally proposed several practical suggestions for future research, which provided directions and guidance toward further product development and process industrialization.