The aggregation of amyloid proteins is highly related to the occurrence and development of neurodegenerative and metabolic diseases. The detection of amyloid fibrils or monitoring fibrillation process would be necessary to understand the fundamental knowledge about the diseases and further facilitate the research for the drug discovery and disease treatment. In this study, three proto-berberine alkaloids, i.e. berberine, palmatine and coptisine, were examined as three distinctive fluorescent probes to detect amyloid fibrils. These three alkaloids were found to be sensitive to the microenvironment, i.e. viscosity and polarity, with varied fluorescence intensity. They could sensitively probe insulin and lysozyme fibrils with turn-on fluorescence, but did not respond to protein monomers, merited with advantages of larger Stokes shift, greenish-yellow fluorescence and no interference with the fibrillation process. Hydrophobic, electrostatic and hydrogen bond interactions were explored to exist between alkaloids and the fibrils. Moreover, these alkaloids succeeded in monitoring the aggregation process of amyloid proteins in vitro and imaging the fibrils in living cells. The present study demonstrates that the three alkaloids could be the potential candidate fluorescent probes for amyloid fibrils. CI - Copyright (c) 2023 Elsevier B.V. All rights reserved.