<p>Multiplexed nucleic-acid detection is essential for molecular diagnostics and spatial genomics, but conventional fluorescence methods are often limited by spectral overlap, nonspecific signals, and restricted encoding capacity. We present a spatial fluorescence barcode (SFB) platform based on transiently luminescent DNA beads (TLDBs) that enables single-color, high-plex readout. In this method, targets are encoded through the spatial arrangement of DNA-functionalized beads, eliminating the need for multicolor labeling or spectral unmixing. Target recognition is achieved through toehold-mediated strand displacement, and built-in nucleases enable autonomous enzymatic resetting for repeated use of probes. The system employs monochromatic spatial encoding, decoupling encoding capacity from spectral channels, and features a simplified probe design and decoding workflow. Self-resetting probes not only streamline the encoding process but also enhance practicality by allowing repeated assays without the need to re-prepare costly probe combinations. We demonstrate robust detection of pathogen-derived nucleic acids in infected blood and cancer-associated microRNAs in tissue samples, validating the platform’s clinical applicability. Compared to existing barcoding strategies, SFB integrates monochromatic spatial encoding, simplified design, and autonomous reusability, offering a practical, scalable, and cost-effective solution for high-throughput nucleic acid analysis.</p>