Microfluidics represents a viable platform for performing high throughput screening (HTS) because of its ability to automate fluid handling and generate fluidic networks with high number densities over small footprints appropriate for the simultaneous optical interrogation of many screening assays. While most HTS campaigns depend on fluorescence, readers typically use point detection and serially address the assay results significantly lowering throughput or detection sensitivity due to a low duty cycle. To address this challenge, we present here the fabrication of a high-density microfluidic network packed into the imaging area of a large field-of-view (FoV) ultrasensitive fluorescence detection system. The fluidic channels were 1, 5 or 10 μm (width), 1 μm (depth) with a pitch of 1-10 μm and each fluidic processor was individually addressable. The fluidic chip was produced from a molding tool using hot embossing and thermal fusion bonding to enclose the fluidic channels. A 40× microscope objective (numerical aperture=0.75) created an FoV of 200 μm, providing the ability to interrogate ∼25 channels using the current fluidic configuration. An ultrasensitive fluorescence detection system with a large FoV was used to transduce fluorescence signals simultaneously from each fluidic processor onto the active area of an electron multiplying charge-coupled device. The utility of these multichannel networks for HTS was demonstrated by carrying out the high throughput monitoring of the activity of an enzyme, apurinic Endonuclease 1, used as a model-screening assay.