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Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re < 1. Moreover, we achieved continuous focusing of live cells with different sizes ranging from 10 to 15 µm, i.e. human T-cell lymphoma Jurkat cells, rat adrenal pheochromocytoma PC12 cells and dog kidney MDCK cells. The glass grooves fabricated by fs laser are expected to be integrated with on-chip detection components, such as contact imaging and fluorescence lifetime-resolved imaging, for various biological and biomedical applications, where particle focusing at a relatively low flow rate is desirable.