Asphodelus-based CQDs were successfully synthesized in a single step by the hydrothermal synthesis method for detection of β-carotene. TEM, XRD, FTIR, XPS, Zeta potential, UV–Vis absorption and fluorescence measurements were taken to structurally and optically characterize the newly synthesized CQDs. It was determined that the characterized CQDs show high selectivity and sensitivity only to β-carotene among 11 different biomolecules. The detection limit of CQDs for β-carotene was calculated as 8.28 µM. It was determined that the new CQDs respond very quickly and stably without interfering with any other biomolecules. The studies in different pH environments exhibited that CQDs can detect β-carotene at all pH studied. Real sample experiments show that β-carotene can be determined accurately and reliably in practical applications with the novel CQDs. It was thought that the results obtained from this study will make significant contributions to the studies on β-carotene detection, which are limited in the literature.