TiO2 nanoparticles are highly stable, eco-friendly in nature, having low cost, act as a photo catalyst also having antimicrobial properties. Considering the effect of TiO2 nanoparticles on seeds, a study was conducted during March 2022 to June 2022 at Department of Genetics and Plant Breeding, SHUATS, Prayagraj (U.P). In this study onion seeds of variety Nasik Red N-53 were collected to investigate the effect of TiO2 nanoparticles on the seedling characters as well as on the biochemical characters under storage in ambient conditions. Onion seeds were treated with different concentration of TiO2 nanoparticles (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 and 140 ppm) along with control and stored in two containers; viz. tin container (C1) and aluminum foil pouch (C2). All the seedling parameters were evaluated every month during storage. The experiment was conducted in factorial CRD with 4 replications. The experimental result showed that the storage containers influenced the seedling characters of onion. Seeds stored in aluminum foil pouch (C2) exhibited highest germination per cent (52.93 %), speed of germination (3.76), root length (2.27 cm), shoot length (4.11 cm), seedling length (6.38 cm), fresh weight (0.176 gm), dry weight (0.0205 gm), seed density (1.025 gm/cm3), dehydrogenase activity (0.213 OD/g mL), catalase activity (0.0220 nmol/min/mg protein) and exhibited lowest moisture per cent (8.05 %) and lowest electrical conductivity (0.970 dS/m) as compared to tin container at the end of 3 months of storage. Seed treated with TiO2 nanoparticles @40ppm (T4) for 2 hours performed better in terms of seedling parameters; viz. germination per cent (58.5 %), speed of germination (4.20), root length (3.06 cm), shoot length (4.58 cm), seedling length (7.64 cm), fresh weight (0.222 gm), dry weight (0.0259 gm), seed density (1.056 gm/cm3), dehydrogenase activity (0.337 OD/g mL), catalase activity (0.0375 nmol/min/mg protein) and recorded lowest moisture per cent (8.04 %) and lowest electrical conductivity (0.951 dS/m) as compared to control after 3 months of storage. The study concluded that seed treated with TiO2 @40 ppm and stored in aluminum foil pouch can be used to expand the storability of onion seeds under ambient condition.