The gentle separation mechanism has made field-flow fractionation particularly suited to samples of biotechnological interest, from proteins and nucleic acids to viruses, subcellular units and whole cells. Recent progress in field-flow fractionation technology, as well as the development of coupled techniques combining field-flow fractionation capabilities with the specificity and sensitivity of well-established analytical methods, opens up new biotechnological applications for field-flow fractionation. The most recent appealing applications include: sorting and fingerprinting of bacteria for whole-cell vaccine production; noninvasive and tagless sorting of immature and stem cells; separation of intact proteins and enzymes in top-down proteomics; and the development of flow-assisted, multianalyte immunoassays using nano- and micron-sized particles with immobilized biomolecules.