Wolfberry (Lyciumbarbarum) is a typical traditional Chinese medicinal and edible fruit, sharing a variety of ingredients and functional activities, such as anti-aging, immune regulation, and anti-atherosclerosis. One of the wolfberry cultivars, Hubei hybrid wolfberry was introduced from the doubled Ningxia wolfberry that crossed with the local wild one in Enshi Prefecture, Jianshi County of Hubei Province in China. A previous study found that this new type of wolfberry was not suitable to process into the conventional dried products, due mainly to higher moisture content, compared with the original Ningxia wolfberry. Therefore, it is highly urgent to develop a new processing approach for the wolfberry products, further improving the conversion rate of Hubei hybrid wolfberry. Alternatively, a characteristic fruit fermentation using lactic acid bacteria can provide a unique flavor product, while transform the types and increase the content of active substances for high nutritional value and health benefits. However, only a few reports were focused on the effects of lactic acid bacteria fermentation on the nutritional quality of wolfberry juice. Taking Hubei hybrid wolfberry as raw material, this study aims to investigate the physicochemical properties, main active components, and antioxidant activity in vitro of wolfberry juice before and after lactic acid bacteria fermentation. 6 kinds of lactic acid bacteria were selected (Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus fermentum) for fermentation. A principal component analysis was utilized to evaluate the quality of fermented wolfberry juice, where the most suitable lactic acid bacteria strain was obtained for wolfberry juice fermentation. The results showed that 6 kinds of lactic acid bacteria grew well in wolfberry juice, where the viable count reached above 10.0 lg CFU/mL. The total contents of sugar and reducing sugar in the juice were significantly reduced (P <0.05) after fermentation. A high capacity of acid production was achieved in the Lactobacillus plantarum and Streptococcus thermophilus, where the total acid contents were 6.74 and 6.07 g/kg, respectively. The total phenol content in wolfberry juice fermented by Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus rhamnosus, and Lactobacillus fermentum increased by 13.76% to 28.07%, compared with unfermented wolfberry juice. Nevertheless, there was no significant difference in the content of total phenols in the wolfberry juice fermented by Lactobacillus acidophilus and Lactobacillus casei (P>0.05). The total flavonoid content increased by 55.80% to 161.97% after fermentation. The antioxidant activities of fermented wolfberry juice were also significantly improved (P<0.05). Correlation analysis showed that the increase in antioxidant activity was closely related to the content of total phenols and total flavonoids. Three principal components were extracted in a principal component analysis, covering three levels of fermentability, nutritional quality, and functionality, indicating a higher quality of fermented wolfberry juice. The cumulative variance contribution rate was 82.344%. The comprehensive score ranking demonstrated that the quality of wolfberry juice fermented by Lactobacillus plantarum and Lactobacillus fermentum was better, suitable for a starter for green processed beverages of Hubei hybrid wolfberry.