The illustration of the correlation between lipid droplets (LDs) variation and nonalcoholic fatty liver disease (NAFLD) is a challenging and important work in biomedical research. Herein, a red emission fluorophore LD-HW containing donor-π-bridge-acceptor (D-π-A) structure was readily constructed and systematically investigated. It was found that LD-HW could selectively identify polarity variation accompanying with an obvious blue-shift (around 80 nm) in fluorescence spectra, and a sharp enhancement (about 440-fold) in fluorescence quantum yield (QY) over the solvent polarity ranging from water (polarity parameter Δf = 0.3200) to 1,4-dioxane (Δf = 0.0205). In addition, probe LD-HW could precisely light up LDs within a short time (≤5 min) through a wash-free procedure and real-time monitor the dynamic behavior of cellular LDs. More importantly, LD-HW exhibited an excellent performance in differentiating fatty liver through in vivo imaging the change of cellular LDs. The in situ fluorescence spectra of corresponding tissue section proved that polarity level in the liver of NAFLD mice was lower than that in normal mice. Taken together, probe LD-HW presented great potential in non-invasive diagnosis of fatty liver through in vivo imaging.