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Micro flow cytometer has been widely used for counting and identifying individual cells. This study presents a novel microfluidic device for arranging cells/particles to a single file in the vertical direction for obtaining a great spatial resolution of signal detection in a micro-flow cytometric chip. Using the isotropic wet etching process and the photomask design, a sequence of micro-weir structures with cascaded height was fabricated on both top and bottom of the micro-fluidic channel. The experimental result shows that the fluorescence-labeled particles with the diameters of 7 and 15 μm can be successfully discriminated using this proposed cascaded microweirs. The calculated coefficients of variation for the signal distribution of these two particles are 29.4% and 14.6%, respectively, indicating significantly enhances the detection performance. Unlike the conventional focusing techniques in vertical direction, the proposed cascaded micro-weirs can provide a simple yet effective flow focusing in vertical direction. The particles of different sizes can be validly arranged to a single file for providing a high-precision counting and discrimination capacity in micro-flow cytometric detection.