This study is aimed to produce and characterize a novel gluten-free ingredient from oat through sprouting at 18 °C for 96 h. The nutritional and bioactive properties as well as key enzymatic activities were studied in sprouted oat powder and compared with those of oat grain powder (control). Sprouted oat powder was an excellent source of protein (10.7%), β-glucan (2.1%), thiamine (687.1 μg/100 g), riboflavin (218.4 μg/100 g), and minerals (P, K, Mg and Ca), and presented better amino acid and fatty acid compositions and levels of γ-aminobutyric acid (54.9 mg/100 g), free phenolics (507.4 mg GA/100 g) and antioxidant capacity (1744.3 mg TE/100 g) than control. Enhanced protease and α-amylase and reduced lipase activities were observed in sprouted oat powder, which are promising features to improve its nutritional, sensorial and health-promoting properties. These results support the use of sprouted oat powder as a promising gluten-free functional ingredient.