Fluorescent nanostructures based on carbon, or carbon dots, are attracting much attention and interest because of their diverse properties which can be applied in several fields of knowledge, such as optics, biomedicine, environmental research, among others. Such properties are in part, derived from its intrinsic luminescence from tunable functional groups. In this work, we produced carbon nanodots (CND) using agro-industrial residues, such as Lolium perenne and malt bagasse. The methods used were conventional hydrothermal syntheses and microwave-assisted hydrothermal synthesis. To the best of our knowledge, this is the first time that carbon dots synthesized from this ryegrass type are reported. The synthesis methods were one step (no catalyst, base, or acid were added for passivation), and the functional groups responsible for the luminescence and high solubility in water were identified by infrared spectroscopy, being mainly C=O, C–OH, C–N, and N–H. According to our theoretical studies, the C=O group introduced a new energy level for electronic transitions that can affect the emission properties. Fluorescence images of osteoblasts using CNDs were acquired and their chelating property towards Pb2+ and Cr6+ detection was tested.