In the liquid state, UV-visible and fluorescence spectroscopy was used to examine the inclusion complexes of nortriptyline (NP) and β-cyclodextrin (β-CD). The degree of inclusion complexation causes NP’s absorbance and fluorescence intensity to be significantly increased during interaction with β-CD. The binding constant was determined by UV-VIS and fluorescence spectroscopy, and the results indicated a 1:1 stoichiometry for the inclusion complex at 303 K. Complexation is a spontaneous and exothermic process, as determined by Gibbs’s free energy change. To produce solid inclusion complexes (ICs), mixing and co-precipitation were used, which were then characterized using Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), X-ray powder diffraction (XRD), and thermogravimetric analysis/differential scanning calorimetry (TGA/DSC). According to molecular docking studies, the aromatic ring of the NP does not penetrate the secondary hydroxyl rim of the β-CD cavity, but the aliphatic part of the NP trapped in the cavity is more thermodynamically advantageous. NP and NP: β-CD-ICs were screened for in vitro cytotoxicity on Michigan Cancer Foundation-7 (MCF-7) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and the results showed that the cytotoxicity was not affected by creating an ICs.