AbstractOptical-based microfluidic cell sorting has become increasingly attractive for applications in life and environmental sciences due to its ability of sophisticated cell handling in flow. The majority of these microfluidic cell sorting devices employ two-dimensional fluid flow control strategies, which lack the ability to manipulate the position of cells arbitrarily for precise optical detection, therefore resulting in reduced sorting accuracy and purity. Although three-dimensional (3D) hydrodynamic devices have better flow-focusing characteristics, most lack the flexibility to arbitrarily position the sample flow in each direction. Thus, there have been very few studies using 3D hydrodynamic flow focusing for sorting. Herein, we designed a 3D hydrodynamic focusing sorting platform based on independent sheath flow-focusing and pressure-actuated switching. This design offers many advantages in terms of reliable acquisition of weak Raman signals due to the ability to precisely control the speed and position of samples in 3D. With a proof-of-concept demonstration, we show this 3D hydrodynamic focusing-based sorting device has the potential to reach a high degree of accuracy for Raman activated sorting.