This report proposed a promising method that combined microfluidic techniques, phase separation and selective dissolution to prepare uniform concave polystyrene (PS) microparticles. Monodisperse emulsion droplets containing PS and poly(methyl methacrylate) (PMMA) in chloroform were firstly obtained using a microcapillary-based microfluidic device. Uniform PS/PMMA Janus composite particles were then generated through the phase separation between PS and PMMA within the emulsion droplets during organic solvent evaporation. Subsequently, uniform PS concave particles were obtained through selective dissolution of the PMMA domain in the Janus composite particle with acetic acid. Moreover, depletion-mediated directional self-assembly of the prepared concave PS particles was investigated, and polymeric worm-like chains with head-to-tail configuration were observed.