This study introduces a novel approach to single-molecule fluorescence multiplexing using a set of composite fluorescent labels called FRETfluors. FRETfluors are nanostructured constructs composed of DNA, Cy3, and Cy5, designed to exhibit tunable spectroscopic properties through variations in geometry, fluorophore attachment chemistry, and DNA sequence. The authors demonstrate the labeling and detection of low-concentration mixtures of mRNA, dsDNA, and proteins using an anti-Brownian electrokinetic trap (ABEL trap). The ABEL trap enables high-precision multi-parameter identification of labeled biomolecular targets, including brightness, fluorescence lifetime, anisotropy, and emission spectrum. By combining these parameters, the authors achieve robust classification of FRETfluors in mixtures, selecting a subset of 27 FRETfluors for multiplexed detection. This approach allows for the multiplexing of up to 27 labels, significantly expanding the capabilities of single-molecule fluorescence multiplexing. The study also highlights the potential for wash-free labeling and detection of diverse biomolecular targets, demonstrating the versatility and sensitivity of the FRETfluor system.This study introduces a novel approach to single-molecule fluorescence multiplexing using a set of composite fluorescent labels called FRETfluors. FRETfluors are nanostructured constructs composed of DNA, Cy3, and Cy5, designed to exhibit tunable spectroscopic properties through variations in geometry, fluorophore attachment chemistry, and DNA sequence. The authors demonstrate the labeling and detection of low-concentration mixtures of mRNA, dsDNA, and proteins using an anti-Brownian electrokinetic trap (ABEL trap). The ABEL trap enables high-precision multi-parameter identification of labeled biomolecular targets, including brightness, fluorescence lifetime, anisotropy, and emission spectrum. By combining these parameters, the authors achieve robust classification of FRETfluors in mixtures, selecting a subset of 27 FRETfluors for multiplexed detection. This approach allows for the multiplexing of up to 27 labels, significantly expanding the capabilities of single-molecule fluorescence multiplexing. The study also highlights the potential for wash-free labeling and detection of diverse biomolecular targets, demonstrating the versatility and sensitivity of the FRETfluor system.