Olive pits, a waste product derived from olive oil production, have a high carbohydrate content attractive for biorefineries. Nanotechnology, making possible the preparation of nontoxic carbon-based nanoparticles, namely, carbon dots, provides a compelling opportunity for the valorisation of such waste feedstocks with enormous advantages. In this work, biomass was subjected to a soft one-step treatment in which trifluoroacetic acid and different solid catalysts were used. Fluorescent nanosized (4.5 nm) particles were obtained, displaying the characteristic behavior of carbon dots. These nanoparticles exhibited a fluorescence emission at 500 and 602 nm when treated with the solid catalyst and trifluoroacetic acid, respectively. XPS and NMR analyses showed oxygen-rich content on the surface of the carbon particles which could explain a proton transfer in the excited state (ESIPT effect). Studies to evaluate the effects of synthesized nanoparticles on HEK 293T cells were also performed, showing their biocompatibility. © Copyright 2019 American Chemical Society.