This study introduces FRET-fluors, a set of fluorescent labels designed for single-molecule multiplexed spectroscopic detection. FRET-fluors are constructed from three chemical components (DNA, Cy3, and Cy5) and are engineered to have tunable spectroscopic properties through variations in geometry, fluorophore attachment chemistry, and DNA sequence. The labels enable the identification of unique spectroscopic signatures for each FRET-fluor, allowing for the differentiation of bound from unbound labels in a wash-free sensing approach. The inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis, complementing changes in FRET efficiency, thereby increasing the number of distinguishable labels while maintaining chemical compatibility. The study demonstrates the use of an anti-Brownian electrokinetic (ABEL) trap for detecting low-concentration mixtures of mRNA, dsDNA, and proteins with high precision. The ABEL trap enables multi-parameter identification of labeled biomolecules at sub-picomolar concentrations. The FRET-fluors were tested in various combinations, and statistically optimal subsets were selected for multiplexing applications, with up to 27 FRET-fluor labels demonstrated. The results show that small changes in dye photophysics can produce uniquely identifiable spectroscopic signatures, and that FRET-fluor structures may partially protect the cyanine dyes against interactions with target molecules or solvent. The study also shows that FRET-fluors can be used for sequence-specific labeling of mRNA, dsDNA, and proteins, with the ability to distinguish between bound and unbound labels. The FRET-fluors were tested in both simple and complex mixtures, demonstrating their effectiveness in detecting low-abundance biomarkers. The results indicate that FRET-fluors can be used for the characterization of dilute, highly heterogeneous mixtures of different types of biomolecules. The study concludes that FRET-fluors offer a promising approach for single-molecule spectroscopic multiplexing, with the potential for further development and application in various fields.This study introduces FRET-fluors, a set of fluorescent labels designed for single-molecule multiplexed spectroscopic detection. FRET-fluors are constructed from three chemical components (DNA, Cy3, and Cy5) and are engineered to have tunable spectroscopic properties through variations in geometry, fluorophore attachment chemistry, and DNA sequence. The labels enable the identification of unique spectroscopic signatures for each FRET-fluor, allowing for the differentiation of bound from unbound labels in a wash-free sensing approach. The inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis, complementing changes in FRET efficiency, thereby increasing the number of distinguishable labels while maintaining chemical compatibility. The study demonstrates the use of an anti-Brownian electrokinetic (ABEL) trap for detecting low-concentration mixtures of mRNA, dsDNA, and proteins with high precision. The ABEL trap enables multi-parameter identification of labeled biomolecules at sub-picomolar concentrations. The FRET-fluors were tested in various combinations, and statistically optimal subsets were selected for multiplexing applications, with up to 27 FRET-fluor labels demonstrated. The results show that small changes in dye photophysics can produce uniquely identifiable spectroscopic signatures, and that FRET-fluor structures may partially protect the cyanine dyes against interactions with target molecules or solvent. The study also shows that FRET-fluors can be used for sequence-specific labeling of mRNA, dsDNA, and proteins, with the ability to distinguish between bound and unbound labels. The FRET-fluors were tested in both simple and complex mixtures, demonstrating their effectiveness in detecting low-abundance biomarkers. The results indicate that FRET-fluors can be used for the characterization of dilute, highly heterogeneous mixtures of different types of biomolecules. The study concludes that FRET-fluors offer a promising approach for single-molecule spectroscopic multiplexing, with the potential for further development and application in various fields.