We present the application of wavefront sensing to three-component, three-dimensional micro particle tracking velocimetry (µPTV). The technique is based upon examining the defocus of the wavefront scattered by a tracer particle and from such information establishing the 3-D tracer location. The imaging system incorporates a cylindrical lens acting as an anamorphic element that creates different magnifications in the two orthogonal axes. A single anamorphic image is obtained from each tracer, which contains sufficient information to reconstruct the wavefront defocus and uniquely identify the tracer's axial position. A mathematical model of the optical system is developed and shows that the lateral and depth performance of the sensor can be largely independently varied across a wide range. Hence, 3-D image resolution can be achieved from a single viewpoint, using simple and inexpensive optics and applied to a wide variety of microfluidic or biological systems. Our initial results show that an uncertainty in depth of 0.18 µm was achieved over a 20-µm range. The technique was employed to measure the 3-D velocity field of micron-sized fluorescent tracers in a flow within a micro channel, and an uncertainty of 2.8 µm was obtained in the axial direction over a range of 500 µm. The experimental results were in agreement with the expected fluid flow when compared to the corresponding CFD model. Thus, wavefront sensing proved to be an effective approach to obtain quantitative measurements of three-component three-dimensional flows in microfluidic devices. © 2009 Springer-Verlag.