This paper details a three-dimensional sheath flow sorting microsystem for separation and collection of biomolecules. High-speed particle and biomolecule 3D sheath flow sorting system was realized using reversible thermosensitive hydrogel. The sol–gel transfer of the thermosensitive hydrogel generated by focused IR laser irradiation performs flow switching, the response time of the sol–gel transformation using Mebiol Gel™ was less than 1 ms. For actual bio-applications, accurate and high-speed micro sorter is realized by optimizing the channel structure and by utilizing 3D sheath flow. High-speed sorting less than 3.0 ms from detection to separation of targets and no error sorting for 45 min (100,000 counts) in 3D sheath flow sorting system was realized. 3D sheath flow can be focused to 1.2 μm of transverse distribution and 6.0 μm of longitudinal distribution in regard to microbeads of sample flow. The same flow velocity and flow line of the focused sample flow are kept at constant laser irradiation time and power to sort the target. Since sorting was performed in simple and disposable PDMS-glass microchannels without any electric stimulation and mechanical valve structures, the proposed system is suitable for many biochemical applications.