The charge transfer complex (CT) chemistry is studied between electron donor 6-Aminoindole (AMI) and electron acceptor Picric acid (PA) at 25 °C, in an acetonitrile medium by using UV–Visible absorption spectroscopy. The molecular ratio of the CT complex was determined to be 1:1 by using jobs variation and spectrophotometric titration processes. To assess the stability constant (KCT), and molar extinction coefficient (ƐCT) by using the Benesi-Hildebrand equation. These outcomes reveal that the synthesized CT complex is highly stable. The thermodynamic study is carried out in different temperatures (20-45 °C), and these results indicate increases the stability with increasing temperature. The CT complex structure was analyzed by FT-IR, 1H NMR, and 13C NMR. The surface morphology and elemental composition were identified by the SEM-EDX technique. The crystalline nature was analyzed by powdered XRD investigation. The CT complex has been screened for DNA binding and invitro cytotoxicity. The DNA binding constant value (2.3 × 105 M−1) and in vitro cytotoxicity test results on HeLa, suggest that it could be used as a potential medicinal drug in the future. Finally, the geometry optimizations were carried out by the DFT with the wB97XD method and 6-31+G(d,p) basis set. Here we investigated different parameters like bond parameters, NBO analysis, FMO analysis, DOS, PDOS, and computed electronic spectra. The computational analysis provided additional support for the experimental CT complex formation.