Gold nanoclusters (AuNCs) have emerged as promising tools for biomedical and environmental applications due to their photoluminescence, biocompatibility, and molecule-like electronic structure. This study presents a novel AuNC-based sensor platform, characterized by eco-friendly synthesis, label-free functionality, and ultrasensitivity for biomolecular detection. AuNCs were synthesized using a green chemistry approach without toxic solvents, yielding strong optical properties with an absorbance peak at 400 nm and emission at 600 nm. Functionalization with thiolated single-stranded DNA (ssDNA) enabled fluorescence-based detection of specific DNA sequences with a limit of detection in the attomolar range. The sensor demonstrated high specificity, distinguishing target DNA from non-specific sequences in both buffer solutions and complex biological matrices, including blood. The modular design allows adaptation to detect various biomolecules by incorporating specific aptamers. This versatile, cost-effective platform combines eco-friendly synthesis, high sensitivity, and specificity, offering significant potential for advanced diagnostics and environmental monitoring in real-world settings.