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Paper-based diagnostic assay is quite popular in point-of-care testing (POCT). In this work, we reported a series of hybrid fluorophores based on fluorescein derivatives and investigated their fluorescent properties in detail. To extend the practical application of these hybrid fluorophores, we further constructed the corresponding probes (FN1-FN4) for Cu2+ detection. In comparison with the other three probes, FN4 showed high selectivity and sensitivity, as well as response to Cu2+ within 20 min. Particularly, by introducing this probe into the paper-based device, we could achieve simple, high-throughput, visual and highly sensitive platform for POC detection of Cu2+. Finally, we assessed the paper-based device in real samples such as serum, cytochylema and lake water to demonstrate its promising application. As a result, satisfied performance for Cu2+ detection was achieved, which indicates the great potential of our strategy in the construction of POCT system for detecting analytes. Image 1 • Novel hybrid fluorophores based naphthofulorescein dyes have been synthesized to show excellent fluorescence properties. • Hybrid fluorophores-based paper device for visual detection of Cu2+ has been developed. • Suitable for point-of-care detection, the paper device could visualize high-throughput Cu2+ detection in real samples. [ABSTRACT FROM AUTHOR]