3-Deoxyanthocyanidins (3-DAS) are the unique flavonoid compounds in sorghum. The majority of research to date have concentrated on the biological activity and extraction of 3-DAS from sorghum and lacked systematic purification and identification investigations. Herein, a facile method for the purification of sorghum 3-DAS from the acidic methanol solution crude extract using macroporous resins (MARs) was proposed and investigated in this work. Based on the experimental data, commercial MAR AB-8 was selected to achieve the purification of sorghum 3-DAS. The adsorption-desorption experiment confirmed the optimal procedure. With this procedure, the total purity of 3-DAS was 19.32%, and 3-DAS content in the purified product was 8.4 times higher than that in the crude extract. UPLC-QTRAP-MS/MS identification indicated that the purified 3-DAS product was mainly composed of apigeninidin, luteolinidin, and their methoxy derivatives. Besides, the purified product exhibited better bioactivities than the crude extract, including the scavenging activities for DPPH, ABTS+, and hydroxyl radicals, the total antioxidant capacity, and the inhibitory effects for α-amylase, α-glucosidase, and lipase. This work provides a potential way for the large-scale purification of bioactive sorghum 3-DAS from crude extract, which is significant to deepen the applications of sorghum 3-DAS in the food nutrition and pharmaceutical industries.