A benzo[b]quinolizinium-benzo-15-crown-5 ether conjugate 2a is presented that enables the fluorimetric detection of Mg²⁺ and DNA by a significant light-up effect, along with a change of the emission wavelength with different analytes (Mg²⁺: 495 nm; DNA: 550 nm). The mechanism of the excited-state deactivation of 2a was investigated by steady-state fluorescence spectroscopy in media of varied viscosity and compared with the photophysical properties of methoxyphenyl-substituted benzo[b]quinolizinium 2b (m,p-diOMe), 2c (m-OMe), and 2d (p-OMe) as reference compounds. Compounds 2a–c, which share the m-alkoxyphenyl substituent as the common feature, have low emission quantum yields (Φ_F < 10⁻² in water) but exhibit a significant increase of their fluorescence intensity in viscous glycerol solutions. In contrast, the viscosity of the medium does not influence the emission properties of the parent phenyl-substituted benzo[b]quinolizinium 2e and of the p-methoxyphenyl-substituted derivative 2d. Based on these observations it is concluded that the excited-state deactivation in 2a–c is mainly due to the rotation of the m-alkoxy group about the C_ar–O bond. The interaction of 2a–c with DNA or Mg²⁺ ions was studied by spectrophotometric titrations and CD spectroscopy. Notably, the association of 2a or 2b with DNA or 2a with Mg²⁺ ions induces a strong fluorescence enhancement (15- and 40-fold for DNA, 450-fold for Mg²⁺), which is rationalized by the suppression of the torsional-relaxation of the alkoxy-substituent in the excited state. Additionally, the cation-induced light-up effect of 2a is selective towards Mg²⁺ ions as compared with other cations such as NH₄⁺, Li⁺, Na⁺, K⁺ and Ba²⁺.