Separating particles from a suspension and sorting particles into different size ranges are important to many chemical, biological, and bioengineering applications. In this article, a novel lateral-flow particle separation device is presented for continuous particle fractionation from suspensions. This device is based on three-dimensional multilayer poly(dimethylsiloxane) microchannels, which can be fabricated by high-yield and low-cost molding and transfer-bonding techniques. By varying the dimensions of the microchannels in each layer, particles in a suspension can be fractionated into specific layers based on their sizes. Particle separation is successfully achieved in sorting polystyrene microbeads of 1, 10, and 45 mu m in diameter into different layers. The yield and selectivity of particle separation can be controlled by device geometries such as channel width and length. This novel continuous-flow particle filtration and separation device is expected to find applications in micrototal analysis systems due to its simple fabrication steps, low cost, and capability of particle separation in a deterministic fashion.