The rise of superbugs, including antibiotic-resistant bacteria, and virus outbreaks, such as the recent coronavirus scare, illustrate the need for rapid detection of disease pathogens. Widespread availability of rapid disease identification would facilitate outbreak prevention and specific treatment. The ARROW biosensor microchip can directly detect single molecules through fluorescence-based optofluidic interrogation. The nature of the microfluidic channels found on optofluidic sensor platforms sets some of the ultimate sensitivity and accuracy limits and can result in false negative test results. Yet higher sensitivity and specificity is desired through hydrodynamic focusing. Novel 3D hydrodynamic focusing designs were developed and implemented on the ARROW platform, an optofluidic lab-on-a-chip single-molecule detector device. Microchannels with cross-section dimensions smaller than 10 μm were formed using sacrificial etching of photoresist layers covered with plasma-enhanced chemical-vapor-deposited silicon dioxide on a silicon wafer. Buffer fluid carried to the focusing junction enveloped an intersecting sample fluid, resulting in 3D focusing of the sample stream. The designs which operate across a wide range of fluid velocities through pressure-driven flow were integrated with optical waveguides in order to interrogate fluorescing particles and confirm 3D focusing, characterize diffusion, and quantify optofluidic detection enhancement of single viruses on chip.