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• Two polarity-sensitive fluorescent probes (NNap-LD-Ⅰ , NNap-LD-Ⅱ) with D-A-D structures were accurately synthesized by a simple synthetic method. • The two fluorescent probes (NNap-LD-Ⅰ , NNap-LD-Ⅱ) exhibit good photophysical properties, such as bright fluorescence, large Stokes shift, and resistance to chemical interference. • Both fluorescent probes (Nap-LD-Ⅰ , Nap-LD-Ⅱ) easily penetrate the cell membrane, specifically localize and aggregate in lipid droplets and lysosomes with low cytotoxicity, and yellow and red fluorescent lipid droplets and lysosomes can be observed. • Both fluorescent probes (NNap-LD-Ⅰ , NNap-LD-Ⅱ) were further investigated for the separate targeting of lysosomes and lipid droplets in HepG2 cells. • Both fluorescent probes (NNap-LD-Ⅰ , NNap-LD-Ⅱ) can be used for imaging of zebrafish. Lipid droplets (LDs) and lysosomes, vital subcellular organelles in cells, played irreplaceable roles in the areas involved in the synthesis of cell membranes, the movement of substances and the transfer of energy. Moreover, the imbalance of LDs could cause the occurrence of certain diseases, such as cardiovascular disease, liver cancer, atherosclerosis and diabetes. LDs and lysosomes interact in some biological processes. In this work, two polarity-sensitive fluorescent probes (NNap-LD-Ⅰ , NNap-LD-Ⅱ) were developed and synthesized by simple 1,8-naphthalimide scaffold modifications, which exhibited large Stokes shifts (173 nm and 190 nm), viscosity, pH stability and interference resistance. Notably, they were biocompatible and showed good targeting for bioimaging. LDs, lysosomes in HepG2 cells and zebrafish could be marked by bright yellow and red fluorescence after incubation with probes (NNap-LD-Ⅰ , NNap-LD-Ⅱ). In addition, HepG2 cells were stained for NNap-LD-Ⅰ and NNap-LD-Ⅱ to monitor the corresponding reduction in the number of LDs in starvation-treated cells. Similarly, the targeting of the probes to the lysosomes was diminished during imaging after the cells were treated with chloroquine (CQ), resulting in a reduction in lamellar fluorescence. These remarkable characteristics suggested that the probes NNap-LD-Ⅰ and NNap-LD-Ⅱ were quite efficient fluorescent probes with good visualization of LDs and lysosomes. [ABSTRACT FROM AUTHOR]