Hydrodynamic focusing behavior is characterized by two fluids coflowing at different velocities inside a micro-flow cytometer. In this study, a two-fluid model has been established to describe the flow transport behavior and interaction of sample and sheath fluids. The analysis treats the sample and sheath fluids as two-dimensional, laminar, incompressible, and isothermal. The theoretical model comprises two groups of transient conservation equations of mass and momentum with consideration of the interfacial momentum exchange. The governing equations are solved numerically through an iterative SIMPLEC algorithm to determine the flow properties. Since the ratio of the sheath velocity to the sample velocity varies from 5 to 70, the predicted focusing width and length are in good agreement with the experimental data in the literature. In addition, the present study explored the hydrodynamic focusing flowfield as well as the pressure drop across a micro-flow cytometer and the time needed for the completion of one focusing event in detail. To enhance the understanding of hydrodynamic focusing in the design of cytometers, ten numerical experiments were conducted to examine the effects of the inner nozzle length, inner nozzle exit width, inner nozzle shape, and fluid properties on the width of the focused sample stream.