Polyploidy breeding is a promising strategy to enhance stress tolerance and functional quality in fruit crops. Tetraploid Vitis amurensis (V. amurensis) may offer superior advantages over its diploid counterpart, including increased biomass, improved antioxidant capacity, and greater adaptability to environmental stress. This study investigated the effects of colchicine concentration and treatment duration on tetraploid induction in Vitis amurensis and analyzed the initial growth characteristics of colchicine-induced tetraploids. Nodal explants were treated with four colchicine concentrations (0.01–0.2%) for three durations (8, 16, and 24 hours), and ploidy levels were determined using flow cytometry. Morphological and physiological traits were evaluated after ex vitro acclimatization. The highest induction efficiency was achieved with 0.1–0.2% colchicine for 16 hours, optimizing tetraploid production while maintaining a 50% survival rate. Higher colchicine concentrations and longer exposure times significantly reduced survival rates, indicating a dose-dependent cytotoxic effect. Tetraploid V. amurensis exhibited a 2.7-fold increase in the average total length of the main shoot and a 1.93-fold increase in stomatal size compared to diploid V. amurensis regardless of genotypes.. Antioxidant properties were markedly improved, with phenolic and flavonoid contents up to 2.5 times greater than in diploids, alongside increased DPPH radical scavenging activity and Ferric reducing Antioxidant power values, reflecting better oxidative stress mitigation. These improvements can be attributed to chromosome doubling, which enhances cellular size, secondary metabolite production, and metabolic efficiency. This study underscores the importance of balancing colchicine efficacy with toxicity for successful tetraploid induction. Tetraploid Vitis amurensis presents strong potential as a climate-resilient and functional grape cultivar, with superior growth and stress tolerance traits. Further in vivo and ex vitro research is needed to confirm these findings and explore mechanisms underlying stress resilience for sustainable viticulture.