Barley grass possesses high nutritional value and antioxidant properties. In this study, the phytochemical constituents and antioxidant enzyme activities in six cultivars of barley grass were explored at three developmental stages: tillering, jointing, and booting stages. Total chlorophyll (Chl t) and carotenoid (Car) content, chlorophyll a/b (Chl a/b) ratio, total nitrogen nutrition (TNN), and total soluble protein (TSP) content, and superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) activities were assayed. The results indicated that the cultivar × development interaction was significant and that developmental stage was the main factor affecting the parameters studied. Cultivars had a negligible effect on these parameters, which varied with the developmental stages. In the tillering stage, Chl t and Car content, TNN, and POD activity achieved their highest value; in the jointing stage, SOD activity peaked; in the booting stage, Chl a/b ratio, TSP content, and PPO activity showed their highest values. TNN showed a negative correlation with TSP. Compared with those in the jointing, Chl t, Car, TSP, TNN content, Chl a/b ratio, and POD and PPO activities increased in the booting and the tillering stages, whereas SOD activity decreased. The differences in phytochemical constituents and antioxidant enzyme activities in barley grass were mainly correlated with the developmental stages. The aim of this study was to demonstrate the influence of developmental stages of barley grass on its phytochemical profile and antioxidant activities. Our results will help understand the mechanism of action of barley grass and provide theoretical support for the therapeutic application of barley grass.