We report on the microfabrication and testing of liquid core waveguides (LCW) using Teflon AF for integration in microfabricated microanalysis systems. Teflon AF has an index of refraction less than that of water allowing it to function as a waveguide when used as a cladding layer surrounding an aqueous core. Straight microchannels (400-μm width, 60-μm depth) etched into Pyrex and soda-lime glass wafers were coated with Teflon AF and sealed with a Teflon AF coated capping wafer. Aqueous fluorescein solutions with varying concentrations were injected into the channels and were illuminated transversely using an ultraviolet light emitting diode. For studying the waveguide attenuation performance, light was focused to a point on the channel. Fluorescence generated in the channel was used to quantify the light collection and waveguide characteristics. The Teflon coating produces a significant enhancement in the amount of light collected in the channel, allowing light to be collected from the 16-mm length tested. This is compared to a control microchannel in glass (no coating), for which the fluorescence drops to the background level in an illumination-detection separation of <4 mm. For sensitivity performance, the entire channel was illuminated. The lower detection limit for spectroscopically resolved fluorescence was ∼10 nMolar.