Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 μM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4–30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98 ± 0.07 × 10⁻⁷ cm²/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.