2,5-Di(hetero)arylthiophenes are key structural scaffolds for several emissive applications and offer tunable fluorescent features attributed to their π-conjugated structures and versatile substituents. In this context, we assessed the photoluminescence of 2,5-di(hetero)arylthiophene derivatives synthesized from 1,3-diynes through cyclization reactions. Through the use of UV‒Vis absorption and steady-state fluorescence emission spectroscopy, we studied the influence of distinct hetero/aryl groups on the fluorescence characteristics of these materials. The integration of experimental and computational data offers a detailed view of the structural and electronic factors governing the photophysical behavior of these compounds. Our findings contribute to a nuanced understanding of the tunability of thiophene-based small molecules, emphasizing the fundamental role of hetero/aryl substituents. These insights have implications for potential applications in optoelectronics and sensor development, positioning these compounds as promising candidates for technological advancements.