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In this work, we have established a simple, efficient, non-toxic, cost-effective and completely green strategy to synthesize carbon dots (CDs) using Mucuna pruriens(alkushi) seeds. Synthesized CDs through facile one-pot hydrothermal route has a spherical shape with an average diameter of 4.02 nm. Surface analysis of these CDs was performed utilizing XPS, FTIR, and zeta potential measurement, confirming the presence of –OH, -NH2, and –COOH groups. The obtained CDs exhibit excellent photostability, high quantum yield and possess outstanding water solubility. Moreover, these CDs provide high response for detection of Au3+ions through fluorescence quenching with a noticeable change in colour of the solution (colorimetric). The LOD was calculated to be 51.20 nM using 3σ/k rule. The mechanism of sensing was established through FTIR, florescence lifetime, UV and Isothermal Titration Calorimetry (ITC). These experiments indicate that a ground state complex formed between CDs and Au3+ions and subsequent reduction to Au0. The specificity towards Au3+ions was tested against different interfering ions and was implemented to analysis the real sample. The synthesized CDs have also the potential to be applied in temperature sensing application. The cytotoxicity and biocompatibility were evaluated against prostate cancer cell line (PC3cell) and a human lung fibroblast cell line (WI38 cell). Finally, Non-cytotoxic CDs were applied for live cell imaging and in vitrosensing of Au3+ions.