This paper reports fluorescein-linked calixarene-based columnar liquid crystals with a broad temperature range of mesophases. The photophysical and density functional theory (DFT) study suggested good electronic behaviour and reactivity of supramesogens. All the synthesized materials (4a–4d) were characterized by FT-IR, ¹HNMR, ¹³C NMR, MALDI-TOF, and elemental analysis and displayed yellow to orange light emission in the solution, thin films, and solid state. Further, all materials showed aggregation-induced emission (AIE) effect with higher quantum yields. The fluorescein-linked supramolecules broadly stabilized the columnar hexagonal phase and formed self-assembly with higher thermal stability. The substituted alkoxy side chains on the terminal position influenced the mesophase temperature range and thermal stability. All the compounds adopted cone conformation, which was confirmed by experimental and computational studies. Further, these fluorescent materials were used to stain model organism nematodes in solution-state aggregation.