We present a new methodology for efficient and high-quality patterning of biological reagents for surface-based biological assays. The method relies on hydrodynamically confined nanoliter volumes of reagents to interact with the substrate at the micrometer-length scale. We study the interplay between diffusion, advection, and surface chemistry and present the design of a noncontact scanning microfluidic device to efficiently present reagents on surfaces. By leveraging convective flows, recirculation, and mixing of a processing liquid, this device overcomes limitations of existing biopatterning approaches, such as passive diffusion of analytes, uncontrolled wetting, and drying artifacts. We demonstrate the deposition of analytes, showing a 2- to 5-fold increase in deposition rate together with a 10-fold reduction in analyte consumption while ensuring less than 6% variation in pattern homogeneity on a standard biological substrate. In addition, we demonstrate the recirculation of a processing liquid using a microfluidic probe (MFP) in the context of a surface assay for (i) probing 12 independent areas with a single microliter of processing liquid and (ii) processing a 2 mm(2) surface to create 170 antibody spots of 50 × 100 μm(2) area using 1.6 μL of liquid. We observe high pattern quality, conservative usage of reagents, micrometer precision of localization and convection-enhanced fast deposition. Such a device and method may facilitate quantitative biological assays and spur the development of the next generation of protein microarrays.