Euglena gracilis (E. gracilis) has recently been attracting attention as a potential renewable source for the production of biofuels, livestock feed, cosmetics, and dietary supplements. Research has focused on strain isolation, productivity improvement, nutrient and resource allocation, and co-product production, key steps that ultimately determine the economic viability and compatibility of the biomass produced. To achieve these characteristics, approaches to select E. gracilis mutants with desirable properties, such as high wax ester content, high growth rate, and high environmental tolerance for biodiesel and biomass production, are needed. Flow-based analysis and sorting can be rapid and highly automated but calls for techniques that can precisely control the position of E. gracilis with varying sizes and shapes in a tightly focused stream in a high-throughput manner. In this work, we use a stepped microchannel consisting of a low-aspect-ratio straight channel and a series of expansion regions along the channel height. We study horizontal and vertical focusing, orientation, rotational, and translational behaviors of E. gracilis as a function of aspect ratio (AR) and channel Reynolds number (Re). By making use of inertial focusing and local secondary flows, E. gracilis with diverse shapes are directed to a single equilibrium position in a single focal stream. As an application of on-chip flow cytometry, we integrate a focusing microchip with a custom laser-two-focus (L2F) optical system and demonstrate the detection of chlorophyll autofluorescence as well as the measurement of the velocity of E. gracilis cells flowing through the microchannel.