Donor acceptor π-π stacking and hydrogen bond mediated self-assembly of two synthetic naphthalene bisimides containing l-amino acids are illustrated. The solution studies exhibit a blue shift in UV/vis spectrum with increasing concentration for both phenylalanine and tyrosine-appended naphthalene bisimides. The single crystal X-ray reveals that both the Phe and Tyr side chains are in the trans position in the reported naphthalene bisimides 1 and 2. In higher order packing, the bisimide 2 molecules self-assemble through intermolecular hydrogen bonds between side chain Tyr -OH and ester -CO, and donor acceptor π-π stacking interactions between the electron deficient central naphthalene moiety and side chain tyrosine ring to generate a staircase architecture. Atomic force microscopy revealed microsphere morphology with diameters of 200-300 nm for bisimides 1 and 2. The microspheres have significant green emission upon excitation at 400 nm. Moreover these two naphthalene bisimides can be used as turn on fluoride sensor in aqueous medium.