Galangin (GAL) is a flavonoid with anti-tumor activity. However, its poor solubility results in low bioavailability thus hindering its clinical application. To overcome this challenge, we have prepared niosomal vesicles by reverse-phase evaporation technique using non-ionic surfactant (Span 20, Span 40, and Span 60) and cholesterol in diverse molar ratios with or without charge inducing agents including Dicetylphosphate (DCP) and Stearylamine (STR). The prepared niosomal formulations were evaluated for drug entrapment efficiency (DEE), Drug loading capacity (DL %), size, and cumulative % drug released. DEE ranged between 45.13 ± 0.35 and 77.69 ± 0.45%. DL% ranged from 9.02 ± 0.51 to 16.72 ± 0.39%. The vesicle size ranged between 173.7 ± 12.6 and 355.6 ± 17.9 nm. The optimized niosomal formulation; F8 (prepared from Span 60, cholesterol, and STR in a 1:1: 0.25 M ratios) was selected depending on its highest DEE, DL%, and suitable drug release profile. F8 was subjected to further investigations as FTIR, DSC analysis, zeta potential, scanning electron microscope, stability studies, and estimation of liver biomarkers. Histopathological examination and immunohistochemical assessment of Minichromosome maintenance 3 (MCM3) were done on extracted liver tissue. Significant improvement of liver biomarkers was observed with galangin loaded niosomes. Histopathological and immunohistochemical examination showed that GAL loaded niosomes exhibit marked decline in MCM3 immunostaining hepatocytes and neoplastic hepatic lesions with a small number of hepatic adenomas, thus GAL loaded niosomes are considered as a promising targeted system for improving anti-tumor activity against liver cancer.