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Refractometry, namely, the measurement of refractive index (RI), provides information about various sample properties such as concentrations and molecular structure. One physical phenomenon which enables precise determination of a sample’s RI in a microscope is the supercritical-angle fluorescence. This effect is observed when the fluorescence from an emitter near a glass-medium interface is captured by an objective lens with a high numerical aperture. The materials’ index mismatch creates a distinguishable transition in the intensity pattern at the back focal plane of the objective that changes proportionally to the RI of the media. Here, we present a refractometry approach in which the fluorophores are preattached to the bottom surface of a microfluidic channel, enabling highly sensitive determination of the RI using tiny amounts of liquid (picoliters). With this method, we attained a standard deviation of 3.1 × 10–5 and a repeatability of 2.7 × 10–5 RI units. We first determine the capabilities of the ...