Spirulina platensis, a microalga known for its bioactive pigments, presents an eco-friendly source of dyes with remarkable optical and fluorescent properties. This study investigates the dual-functionality of Spirulina-derived chlorophyll and phycocyanin as natural dyes for the selective and sensitive detection of biomolecules. The pigments were extracted and analyzed using UV-Vis, FTIR, and photoluminescence spectroscopy, which highlighted their strong interactions with riboflavin (vitamin B2). Both the intrinsic fluorescence and colorimetric responses of the dyes enabled dual-mode detection, with results confirmed using a portable platform. The findings emphasize the significant role of Spirulina-derived compounds in biosensing applications, demonstrating that these natural dyes offer an affordable, sustainable, and effective alternative to synthetic dyes. The dyes’ sensitivity to riboflavin and their ability to function in both fluorescence and colorimetric modes make them promising candidates for various applications, including diagnostics and environmental monitoring. Spirulina-based dyes provide an innovative solution in green sensing technology, prioritizing sustainability without compromising performance. This work contributes to the growing trend of utilizing natural, renewable materials in the development of sustainable sensing platforms, offering a path toward more environmentally friendly biosensing and diagnostic solutions.