Oat is an important crop worldwide, mainly due to its various properties and nutritional status. Recent developments in food nutrition have shown the importance of the different components in oats. Many health benefits of oats attributed to β-glucan and protein have been proven and well-studied over the years. The aim of this project was to investigate the possibility of obtaining high levels of β-glucan and protein fractions from two oat cultivars. Air classification and sieve analysis were used to enrich β-glucan and protein. The effect of classifier wheel speed and particle size on the separation efficiency of β-glucan and protein was investigated. By optimizing the sieving process parameters and air classification process parameters, a normal oat flour fraction whose β-glucan content from 5.1% to 10.1% and protein content from 18.3% to 21.9% could be obtained; a gluten-free oat flour fraction whose β-glucan content from 4.5% to 11.2% and protein content from 16.8% to 22.0% could be obtained. The results showed that the combination of air classification and sieving is a good method to enrich protein and β-glucan from oats.