An aminonaphthalimide-squaraine non-conjugated system was designed and synthetized with the purpose of preparing fluorescent molecule in the 650–700 nm region that could operate via energy transfer (ET) between covalently linked naphthalimide and squaraine chromophores. The photophysical properties of the new fluorescent system were explored with the aim of understanding the ET in one- and two-photon excitation modes. The spectroscopic techniques employed in the characterization includes; absorption, fluorescence, quantum yields and fluorescence lifetime measurements in different solvents. The effect of polarity of solvents on efficiencies of ET were evaluated using one- and two-photon excited fluorescence. The optical behavior of the non-conjugated system was compared with its individual squaraine and naphthalimide moieties. The two-photon absorption (TPA) spectrum of the molecule was obtained between 750 and 1040 nm, with the largest two-photon cross section (δTPA)above 4200 GM. Finally, the applicability of the molecule for fluorescence imaging in the one- and two-photon excitation mode was demonstrated in N13 Microglial cells. The in vitro and in vivo confocal microscopy studies indicated that the non-conjugated system efficiently accumulated in the cytoplasm suggesting it could be utilized as a subcellular probe. Funding for open access charge: Universidad de Málaga / CBUA. This work has been financially supported by MINECO PID2019-104293GB-I00; by Universidad de Málaga-Junta de Andalucía (UMA18-FEDERJA-007), the European Regional Development Fund (ERDF) and ‘‘Plan Propio Universidad de Málaga” (UMA-AndalucíaTECH). European Union’s Horizon 2020 research and innovation programme under EuroNanoMed 3-H2020 DrNanoDAII and MINECO, PROGRAMACION CONJUNTA 2019/PCI 2019-2. The TPA characterization of compounds has been performed by the ICTS ‘‘NANBIOSIS”, more specifically by the U28 Unit of the Andalusian Centre for Nanomedicine & Biotechnology (BIONAND) . This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 713721.