Many fluorophores display interesting features that make them useful biological labels and dyes, particularly in Cell Biology and Cytogenetics. Changes in the absorption-emission spectra (ortho- and metachromasia) are accounted among them. Acridine orange (AO) is one of such fluorochromes with an exemplary orthochromatic vs. metachromatic emission, which depends on its concentration and binding mode to different cell substrates. Here, we revisit the differential AO fluorescence that occurs in selected biological materials, which allows the identification of single-stranded or double-stranded nucleic acids. Although known for a long time, the ultimate reason for this differential phenomenon has not been properly addressed. We propose a potential molecular mechanism that adequately accounts for the distinct AO emission when bound either to denatured or denaturedreassociated DNA. This mechanism, based on theoretical molecular modelling, implies a difference in the degree of overlap of excited state orbitals whenever AO molecules are interacting with bases from single- or doublestranded nucleic acids. In the first case, massive orbital overlapping leads to a metachromatic red AO emission. Otherwise, no excited-state orbital overlapping occurs, due to excessive distance between intercalated AO molecules, which manifests as orthochromatic green fluorescence. Our molecular modelling supports this interplay between orbital overlap/not overlap and metachromatic/orthochromatic fluorescence.