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The ongoing race of biomedical applications has given momentum to the development of graphene quantum dots (GQDs). GQDs are zero-dimensional fluorescent carbon-nanomaterials, with a pronounced quantum confinement effect, and abundant edge states and functional groups. Despite their potential applications, mass-scale synthesis of single crystalline graphene quantum dots (GQDs) with high quantum yields derived via a direct green synthesis approach from bio-wastes is a major challenge. Hitherto, green extract (i.e. sugarcane molasses) driven single crystalline sulphur-doped GQDs (S-GQDs) with a longer decay time, high quantum yield, and excellent biocompatibility have remained unexplored in the bioimaging arena. At the same time, this agro-industrial waste has value in terms of both products and byproducts i.e. zero waste generation resulting in reduced human footprint on the environment. For the first time, we present a facile, large-scale, one-step, economical, template- and catalyst-free synthesis of sustainable, highly crystalline S-GQDs via a hydrothermal route from second generation (2G) bio-wastes. Mechanistic insight into the formation of S-GQDs from their precursor was obtained using powder X-ray diffraction patterns (PXRD). S-GQDs directly obtained from bio-wastes without surface passivation showed the highest quantum yield (QY) ∼ 47% obtained to date. The wide and symmetric emission spectrum of these S-GQDs is instrumental for sensitive detection as labelling nanoprobes. Moreover, their non-toxic behavior, in vitro and in vivo, has a future in quick point-of-care screening and real-time bioimaging. Thus, the as-synthesized bio-waste derived S-GQDs accomplished the purpose of an advanced environmentally friendly and sustainable material which is non-toxic, viable, safe, and cheap. This unprecedented work advances the synthesis of high-quality S-GQDs from bio-waste, which provides a breakthrough in the bioimaging field.