Continuous microfluidic focusing of particles, both synthetic and biological, is significant for a wide range of applications in industry, biology and biomedicine. In this study, we demonstrate the focusing of particles in a microchannel embedded with glass grooves engraved by femtosecond pulse (fs) laser. Results showed that the laser-engraved microstructures were capable of directing polystyrene particles and mouse myoblast cells (C2C12) towards the center of the microchannel at low Reynolds numbers (Re < 1). Numerical simulation revealed that localized side-to-center secondary flows induced by grooves at the channel bottom play an essential role in particle lateral displacement. Additionally, the focusing performance proved to be dependent on the angle of grooves and the middle open space between the grooves based on both experiments and simulation. Particle sedimentation rate was found to critically influence the focusing of particles of different sizes. Taking advantage of the size-dependent particle lateral displacement, selective focusing of micrometer particles was demonstrated. This study systematically investigated continuous particle focusing in a groove-embedded microchannel. We expect that this device will be used for further applications, such as cell sensing and nanoparticle separation in biological and biomedical areas.