The effect of endogenous carbon nanoparticles from food sources is one of the hot topics in the current food research field. The relationship between the foodborne nanoparticle properties and the cytotoxic mechanism has been insufficiently studied. In this work, carbon dots (CDs) with strong fluorescence were found and purified from canned yellow croaker, and their cytotoxicity was investigated for the first time. The canned yellow croaker CDs are nearly spherical with a particle size distribution in the range of 1.8–5.8 nm. The fluorescence quantum yield of the isolated CDs is 9.7% and the maximum excitation wavelength is 340 nm, with a significant redshift phenomenon in fluorescence spectra. The surface elemental analysis showed that the composition of the canned yellow croaker CDs was C (76.42%), N (6.49%), and O (16.7%), and various functional groups are on the surface. The CDs have good stability in sodium chloride solution and the fluorescence intensity was stable within the pH value of 4 to 10. A strong fluorescence quenching effect was found upon the addition of Cu²⁺ and Fe³⁺ to the CD aqueous solution. The CDs can easily enter the interior of the live cells. Moreover, a concentration-dependent behavior of HepG2 cell viability was found when the cells were incubated with the canned yellow croaker CDs. Glycolysis and mitochondrial function analysis of HepG2 cells revealed that both the extracellular acidification rate and oxygen consumption rate significantly decreased in contrast to the normal level prior to the addition of CDs. In addition, the CDs significantly inhibited the glycolytic pathway by reducing the activity of key enzymes hexokinase and pyruvate kinase in the glycolytic pathway.