Capillary-based fluorometry can be considered a promising alternative to cuvette-based methods, enabling low-volume measurements while reducing analyte consumption and waste generation. In this work, we compared the performance of three types of glass container systems, i.e., anti-resonant hollow core fibers, capillaries, and standard cuvettes, using green fluorescent protein (GFP) as a reference analyte. Among these, when the volume of the analyte and the fluorescence intensity are assessed, a capillary accommodating 2.9 μL was found as an optimal choice. The capillary-based interrogation setup was further optimized to enhance sensitivity by adjusting the florescence-exciting laser beam position and optimizing the capillary’s shape. The obtained capillary-based setup achieved a noteworthy 85 % reduction in sample volume compared to previous studies utilizing capillary-based fluorometry, highlighting its significant contribution to material conservation. The system achieved a limit of detection as low as 26 μg/mL (1.01 nM) GFP concentration and a resolution of 3.5 μg/mL. With its low cost and potential for reusability, capillary-based fluorometry presents a compelling alternative to traditional cuvette-based fluorometry, offering a promising solution for micro-volume analysis.