In this work, a core substituted NMI‐conjugated dipeptide (4MNLV) was extensively studied in mixed solvent systems to explore the polarity effect on the self‐assembly pattern and their photophysical property. 4MNLV adopted a J‐ or H‐ type aggregation pattern depending upon the polarity index of the solvent system chosen. The self‐assembly process was achieved through the anti‐solvent effect. UV‐vis study suggested that if the stock solution of 4MNLV was diluted with a relatively more polar solvent (compared to the stock solvent), then the system acquired J‐ type of aggregation pattern by showing a red‐shift in their absorption maxima (λmax). Conversely, when the stock was diluted by a relatively less polar solvent, H‐type of aggregation was observed where blue shift of λmax was noticed. The emission spectra and the lifetime of the self‐assembled materials were also influenced by the chosen solvent system. The chirotopic behaviour of these self‐assembled materials was studied through CD spectroscopy. Morphological study indicated the formation of helical nanofibrillar structures. The bright green fluorescence of these highly biocompatible naphthalimide‐peptide conjugate was used for cell imaging application, indicating its futuristic scope.