Salvia officinalis L. (2n=2x=14) is a perennial plant from Lamiaceae family. It has a long history of medicinal and culinary use. This study was aimed to investigate the polyploidy induction in S. officinalis L. via seed treatment with colchicine concentrations of 0, 0.05, 0.1, 0.25 and 0.5% at 12, 24 and 48 h treatment time as a factorial experiment based on the completely randomized design with three replications. Polyploidy induction firstly detected by morphological, anatomical and physiological characteristics and confirmed by flow cytometry and chromosome observation from leaves. Results showed that the colchicine concentration and treatment duration have significant effects on the survival rate of plants and the percentage of tetraploidy induction. The percentage of survived plants was decreased by increasing the treatment time and colchicine concentration. Meanwhile, the highest percentage of induced tetraploid plants was obtained at 0.25 and 0.5% colchicine with a treatment time of 48 and 24 h, respectively. Tetraploid plants specified by increasing in leaf length, leaf width, plant height, number of leaves, number of nodes, internodes length, stomata size, catalase, peroxidase and polyphenol oxidase activities, total phenolic and flavonoid contents and antioxidant capacity and on the contrary, decreasing in stomata count as compared with diploid ones.