Pyrediyne is one of the recent entries to carbon-based 2D material series with better electronic and charge transport properties compared to graphdiyne. Herein, the preparation of pyrediyne-based quantum dots (PDYQDs), with tunable emission, from nonluminescent 2D precursor pyrediyne is investigated. As-prepared PDYQDs are characterized by various microscopic as well as spectroscopic techniques such as high-resolution transmission electron microscopy, atomic force microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, matrix-assisted laser desorption/ionization, UV-visible, and fluorescence spectroscopy. The simple preparatory method and absence of heavy metal or external passivating agents make this pyrene-containing QDs unique. Moreover, the tunable emission (410–548 nm), notable photoluminescence quantum yield (23%), and negligible cytotoxicity of PDYQDs are explored for bioimaging in MCF7 breast cancer cells. The findings in the present study are encouraging to explore novel carbon-based QDs from their 2D counterparts to enhance their applications in various biological and optoelectronic devices.