We develop microfluidic methods for manipulating and imaging single biopolymers, including DNA and proteins, using flow fields and surfaces. These methods are of importance in the development of microfluidic devices for processing of DNA and other large molecules for genomics applications, and to learn how proteins interact with DNA in cellular processes such as DNA repair, transcription, etc. Using single-molecule optical microscopy and atomic force microscopy experiments combined with Brownian dynamics simulations of DNA molecules, and fluid mechanics analyses, we explore ways in which DNA unraveling can be optimized through manipulation of flow. These methods are applied to analysis of DNA–protein interactions and to understanding the swimming of motile bacteria.