Herein, the synthesis of pyrene conjugated 2,6-di-ortho-tolylpyridine and 2,6-di-para-tolylpyridine structural isomers were achieved e ciently through multicomponent Chichibabin pyridine synthesis reaction. The DFT, TD-DFT and experimental investigations were carried out to investigate the photophysical behaviors of the synthesized novel pyrene-pyridine based isomeric probes. Our studies revealed that, due to the continuous conjugation of the pyrene, pyridine and tolyl moieties, the dihedral angles of the trisubstituents on the central pyridine moiety signi cantly in uences the photophysical properties of the synthesized novel pyrene based uorescent probes. Further, we have comparatively investigated the sensing behaviors of the synthesized tolyl-substituted isomeric ratiometric uorescent probes with metal ions, our studies reveals that both the ortho and para tolyl ratiometric uorescent probes have distinct photoemissive properties in selectively sensing of Hg 2+ ions. Our studies indicates that, the para-tolyl substituted isomer displays more red-shift in wavelength of emission band compared to its ortho isomer analogue during ratiometric uorescent speci c detection of Hg 2+ ions.