In this study, an eco-friendly, cost-effective, and convenient method for preparing biocompatible fluorescent carbon quantum dots (CQDs) by one-pot microwave assisted synthesis from the leaf extract of the medicinal plant Calotropis gigantea, also known as crown flower, has been demonstrated. As-synthesized CQDs demonstrated fluorescence quantum yields up to 4.24 percent. The size distribution of the as-synthesized CQDs varied from 2.7 to 10.4 nm, with a significant proportion of sp2 and sp3 carbon groups verified by nuclear magnetic resonance analysis. The zeta potential of as-synthesized CQDs was measured to be –13.8 mV, indicating the existence of a negatively charged surface with incipient instability in aqueous suspension. Furthermore, as an alternative to organic or synthetic dyes, the development of simple, inexpensive, and non-destructive fluorescence-based staining agents are highly desired. In this regard, as-synthesized CQDs shown remarkable fluorescent staining capabilities in this work and may be utilised as a suitable probe for optical and bio-imaging of bacteria, fungi, and plant cells.