The photophysical properties of three types of hybrid molecules, containing two 1,2,3-triazole isomers with different spatial arrangements and set of surrounding substituents, were investigated. The effect of the heterocyclic scaffold construction and the nature and position of the substituents were revealed by spectral and computational investigations. UV–Vis absorption spectra showed maxima with high molar extinction coefficients. Moderate blue emission (QYs up to 42.2%) and average fluorescence life-time of 1.05–4.53 ns were determined. Positive solvatochromism and intramolecular charge transfer were detected and confirmed by the quantum mechanical calculations. A peculiar effect of a hydrogen bond formation was registered in MeOH, EtOH, i-PrOH, ethylene glycol (EG) solutions and water mixtures. The compounds exhibited an Aggregation Induced Emission Enhancement (AIEE) effect in DMSO-H2O mixtures with a 1.3–3.1 fold increase of QYs and a 1.1–2.8 fold increase of fluorescence life-time. Molecular geometries and electronic characteristics were studied at (Time Dependent) Density Functional Theory ((TD-)DFT) level. The biological behavior of 2H-bis[1,2,3]triazolo)[5,1-a:4′,5′-c]isoquinolines was studied by means of confocal microscopy experiments, which showed that these compounds successfully penetrate the cell membranes, accompanied by bright emission in the cytoplasmic region of the confocal micrographs.