We propose an analytical framework to build a microfluidic microsphere-trap array device that enables simultaneous, efficient, and accurate screening of multiple biological targets in a single microfluidic channel. By optimizing the traps’ geometric parameters, the trap arrays in the channel of the device can immobilize microspheres of different sizes at different regions, obeying hydrodynamically engineered trapping mechanism. Different biomolecules can be captured by the ligands on the surfaces of microspheres of different sizes. They are thus detected according to the microspheres’ positions (position encoding), which simplifies screening and avoids target identification errors. To demonstrate the proposition, we build a device for simultaneous detection of two target types by trapping microspheres of two sizes. We evaluate the device performance using finite element fluidic dynamics simulations and microsphere-trapping experiments. These results validate that the device efficiently achieves position encoding of the two-sized microspheres with few fluidic errors, providing the promise to utilize our framework to build devices for simultaneous detection of more targets. We also envision utilizing the device to separate, sort, or enumerate cells, such as circulating tumor cells and blood cells, based on cell size and deformability. Therefore, the device is promising to become a cost-effective and point-of-care miniaturized disease diagnostic tool.