Abstract <p><i>Viola odorata</i> L., commonly known as sweet violet, is a valuable dicotyledonous plant of the Violaceae family, recognized for its medicinal and ornamental significance. Polyploidy induction using chemical mutagens, such as colchicine, is widely employed to enhance traits in ornamental plants. In this study, four- to six-leaf stage seedlings were treated with colchicine at various concentrations (0–0.5% w/v) for 24, 48, and 72 h via apical meristem immersion to induce tetraploidy. The effects of polyploidization on survival, morphological traits, physiological performance, biochemical composition, phytochemical accumulation, and antioxidant enzyme activities were evaluated. Results showed the highest survival rate (97.78%) in the control group, with 0.3% colchicine for 48 h being the most effective treatment for tetraploid induction. Tetraploid plants displayed typical morphological changes, including increased stomatal size and reduced stomatal density, with cytological analysis confirming chromosome doubling from diploid (2<i>n</i> = 2<i>x</i> = 20) to tetraploid (2<i>n</i> = 4<i>x</i> = 40). Compared to diploids, tetraploids exhibited significantly higher photosynthetic pigments, total protein, phenylalanine ammonia-lyase (PAL) activity, vitamin C, total phenolic, and flavonoid contents, along with enhanced activities of catalase, ascorbate peroxidase, and guaiacol peroxidase. The increased fresh and dry weight in tetraploids further indicates that higher biomass may contribute to the elevated biochemical and phytochemical levels. These findings demonstrate that tetraploid induction effectively enhances morphological, physiological, and biochemical traits, as well as antioxidant capacity in <i>V. odorata</i>, providing a promising strategy to improve its medicinal and ornamental value.</p>