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In this study, a novel radio‐frequency cold plasma‐assisted extraction (CPAE) process was developed for the extraction of anthocyanins from a type of agricultural biomass waste, purple corncob. The effects of cold plasma pretreatment on anthocyanin extraction were investigated, and this process was further optimized using response surface methodology. The components of purple corncob anthocyanins (PCAs) were analyzed by super‐high performance liquid chromatography‐mass spectrum. The antioxidant activity of PCAs was evaluated through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2′‐azinobis‐(3‐ethylbenzthiazoline‐6‐sulfonate) (ABTS) scavenging capacity assays in vitro and physiological behaviors in vivo for Caenorhabditis elegans. The results showed that the optimal conditions of cold plasma pretreatment were discharge power 127 W, working pressure 150 Pa, and the treatment time 75 s. The maximum yield of PCAs was about 4.05 ± 0.05 mg/g corncob, much higher than that of the conventional solvent extraction and ultrasonic‐assisted extraction. PCAs were mainly composed of pelargonidin‐3‐galactoside (66.02 wt.%), cyanidin‐3‐galactoside (25.95 wt.%), cyanidin (8.02 wt.%), and petunidin‐3‐galactoside (0.02 wt.%). The extracted anthocyanins reflected the superior antioxidant activity. In terms of in vitro antioxidant ability, the half maximal inhibitory concentration (IC50) of PCAs for DPPH and ABTS were slightly lower than those of ascorbic acid. Meanwhile, C. elegans exhibited positive effects on several physiological behaviors by feeding PCAs, including a 29.25% increase in lifespan, and increases in antioxidant capacity, superoxide dismutase, and catalase (CAT) activity of 26.28%, 28.93%, and 22.24%, respectively. The CPAE process is therefore a highly competitive candidate for the realization of industrial PCAs extraction, combining some additional advantages of cold plasma treatment such as green process, low cost, short treatment time, and scale‐up production. Practical applications: Agricultural biomass wastes (e.g., corncob, straws, and husks) contain a large number of high value‐added natural active ingredients (e.g., polysaccharides, anthocyanins, and polyphenols). However, a highly efficient extraction of these natural active ingredients is always sought, as the protection of lignocellulosic cell walls hinders the extraction of intracellular products. In this work, the cold plasma‐assisted extraction (CPAE) process showed a comparatively higher anthocyanin yield than the conventional solvent extraction and ultrasonic‐assisted extraction processes. This was due to the fact that the high‐energy helium plasma attack readily broke down the lignocellulosic structure of the plant cell walls, resulting in the rapid release of intracellular purple corncob anthocyanins into the extraction medium. This study suggests that the CPAE process will become an emerging application for the industrial extraction of high value‐added natural active ingredients from agricultural biomass wastes. [ABSTRACT FROM AUTHOR]