<p>Heteroatom-doped carbon dots (CDs) possessing high fluorescent properties make them potential candidates for sensing toxic analytes and heavy metal ions, alongside broad biosensing and imaging applications. In the current work, we report a novel scheme for synthesizing Ulvan-derived CDs co-doped with guanidine isothiocyanate as a nitrogen source with enhanced optical properties. Extraction of the sulfated Ulvan from dried seaweed biomass followed by anion exchange chromatography to isolate fractions with low PDI (polydispersity index) ~ 1.3, which was further used as starter material along with guanidine isothiocyanate as co-dopant led to efficient incorporation of -N within the equitized CDs possessing high fluorescence with quantum yield ~ 0.39. Subsequent bioactive properties of the CDs were inferred for their promising results in cytotoxicity assays, biofilm inhibition, wound scare assay, and biological imaging. Later, the photophysical properties of the CDs were investigated for their selective sensing towards Hg²⁺cations in a pool of other metal ions conferring by a quenching mechanism evident using fluorescence spectroscopy, FTIR, fluorescence decay spectra, and UV spectroscopy studies. Limit of detection (LOD) values and quenching constant values determined as 284&#xa0;nM and 1.68 X 10⁴&#xa0;M⁻¹ further concrete the existence of a static quenching mechanism via stable complex formation between CDs and Hg²⁺ cations. Lastly, CDs recyclability and pH-dependent stability were investigated to prove their potential in real-world applications.</p> Graphical abstract <p></p>