The cost, time, and restrictions on creative flexibility associated with current fabrication methods present significant challenges in the development and application of microfluidic devices. Additive manufacturing, also referred to as three-dimensional (3D) printing, provides many advantages over existing methods. With 3D printing, devices can be made in a cost-effective manner with the ability to rapidly prototype new designs. We have fabricated a micro free-flow electrophoresis (μFFE) device using a low-cost, consumer-grade 3D printer. Test prints were performed to determine the minimum feature sizes that could be reproducibly produced using 3D printing fabrication. Microfluidic ridges could be fabricated with dimensions as small as 20 μm high × 640 μm wide. Minimum valley dimensions were 30 μm wide × 130 μm wide. An acetone vapor bath was used to smooth acrylonitrile-butadiene-styrene (ABS) surfaces and facilitate bonding of fully enclosed channels. The surfaces of the 3D-printed features were profiled and compared to a similar device fabricated in a glass substrate. Stable stream profiles were obtained in a 3D-printed μFFE device. Separations of fluorescent dyes in the 3D-printed device and its glass counterpart were comparable. A μFFE separation of myoglobin and cytochrome c was also demonstrated on a 3D-printed device. Limits of detection for rhodamine 110 were determined to be 2 and 0.3 nM for the 3D-printed and glass devices, respectively.