This study demonstrated an environmentally responsible, inexpensive and simple one-step hydrothermal method to synthesize biocompatible fluorescent carbon quantum dots (CQDs) from Aegle marmelos fruit extract. The near-spherical CQDs were discovered to have a naturally distributed size of 3–6 nm, were unintentionally doped with heteroatoms (Ca, K, and N) without the need for doping element precursors due to mineral contents in extract, had a quantum yield of about 4.06%, strong photostability, and excellent water solubility. Several morphological, compositional, physicochemical, and optical properties were studied. Other variables influencing CQD photoluminescence, such as hydrothermal process parameters and pH sensitivity, were also examined. Zeta potential analysis showed a negative surface charge with a zeta potential value of −5.29 mV and good dispersibility in aqueous solution. Furthermore, as an alternative to organic or synthetic probes, there is a huge need for the development of simple, affordable, and non-destructive fluorescent biomarkers. As-synthesized Ca, K, N-doped CQDs have demonstrated good fluorescent staining abilities and may be employed as a helpful probe for bio-imaging of cancer cells and other organisms. Cell survival and cytotoxicity were found to be satisfactory, with 90% of SH-SY5Y and HCT-116 cells surviving, compared to 63–77% for the other organisms, even at higher dosages (200 µg/mL) for up to 4 days. Display omitted •A facile heteroatom doped CQDs have been synthesized from Aegle marmelos.•Hydrothermal process dependent and pH responsive PL were examined.•Bio-imaging of cancer cells and other living organisms are demonstrated.•Cytotoxicity were found over 90% of SH-SY5Y and HCT-116 cancer cells.