Microfluidics and nanofluidics have recently emerged as analytical tools for the study of biology. These devices have enabled the miniaturization of biological sample preparation and detection methods, toward consuming less sample volume and improving the sensitivity and speed of analysis. This thesis explores methods for rapid detection and sorting of individual biomolecules within a nanofluidic channel. In these devices, constructed using thin-film processing techniques, attoliter-scale volume confinement is formed to isolate individual, fluorophore-labeled biomolecules in solution for absolute quantification. These devices enable studies of the unique attributes of each molecule, often masked in ensemble-averaged measurements. Statistical sampling of many molecules is achieved by voltage-actuated, electrokinetic flow within the nanofluidic device to precisely control molecule analysis rate and achieve high throughput single molecule detection (SMD). This nanofluidic technology is applied to epigenetic analysis, enabling the study of epigenetic modifications at a single molecule level. Viable epigenetic analysis within a nanofluidic device is demonstrated using chromatin, DNA bound with histone proteins, which is shown to remain in its native state during nanofluidic confinement and electrokinetic flow under physiologically-relevant conditions. Detection of an epigenetic modification, DNA methylation, is also demonstrated to elucidate its potential for detecting multiple epigenetic marks on an individual molecule. Subsequently, an architecture for automated, high-speed sorting of individual molecules is developed. In this architecture, digital signal processing methods are implemented in a field programmable gate array to achieve real-time SMD. An electric circuit model is developed to actuate and switch electrokinetic flow of molecules, partitioning them into branches of a bifurcated nanofluidic device. An optical system for parallel SMD is realized to experimentally validate the actuation of molecule sorting in-situ. Combined, these components are utilized in automated, fluorescence-activated sorting of individual, methylated DNA molecules, which were then collected for further analysis. This device is reconfigurable and can be generalized for application to fluorescence-activated separations of other molecule types. Finally, a study of various methods for optofluidic integration is presented. The optical properties of fused-silica, silicon nitride, polydimethylsiloxane, hydrogen silisequioxane, and chemical vapor deposited oxides are investigated to consider their use in SMD applications requiring ultra-low autofluorescence and high confinement of the optical probe volume. Findings were then applied to form an optical waveguide as an fluorescence excitation source toward the dense integration of optical and nanofluidic components. Advisors/Committee Members: Craighead, Harold G (chair), Lovelace, Richard V E (committee member), Kan, Edwin Chihchuan (committee member).