This study interest with formation of the inclusion complex of Neomycin antibiotic with β-cyclodextrin by the colloidal tectonic approach. The new inclusion complex of Neomycin was characterized by Fourier-transformation infrared (FTIR) to confirm the chemical structure of the prepared materials by the shifting that occur in the frequencies. X-ray diffraction (XRD) was investigated. The results show the crystallinity properties of the Neomycin was increased after loading process. The morphology of the prepared Neomycin inclusion complex was examined by Scanning electron microscope (SEM). Moreover, the UV–visible spectroscopy was studied and indicates the optical properties of the Neomycin enhanced after loading. The results of this study established the insertion of Neomycin into the cavity of β-cyclodextrin has been improved the morphology structure, crystallinity and the optical properties of the Neomycin drug.