In this research, a new approach to enzyme inactivation in flour was presented by supercritical technology, considered a sustainable technology with lower energy consumption compared to other technologies that use ultra-high temperature processing. Total protein concentration and the activity of enzymes α-amylase, lipase, peroxidase, polyphenol oxidase, and protease were determined in flour pre-treated with scCO2. During the study, it was observed that the activity of enzymes such as lipase and polyphenol oxidase, was significantly reduced under certain conditions of scCO2 treatment, while the enzymes α-amylase and protease show better stability. In particular, polyphenol oxidase was effectively inactivated below the 60% of preserved activity at 200 bar and 3 h, whereas α-amylase under the same conditions retained its activity. Additionally, the moisture content of the scCO2-treated spelt flour was reduced by 5%, and the fat content was reduced by 58%, while the quality of scCO2-treated flour was maintained. In this regard, the sustainable scCO2 process could be a valuable tool for controlling the enzymatic activity of spelt flour since the use of scCO2 technology has a positive effect on the quality of flour, which was verified by the baking performance of spelt flour with the baked spelt bread as an indicator of quality.