Currently, thermal pasteurisation (TP) remains the most widely applied technique for commercial orange juice preservation; however, a high temperature causes adverse effects on the quality attributes of orange juice. In order to explore a novel non-thermal sterilization method for orange juice, the impacts of thermosonication combined with nisin (TSN) and TP treatments on the quality attributes including microbial and enzyme inactivation and the physicochemical, nutritional, functional, and sensory qualities of orange juice were studied. Both TP and TSN treatments achieved desirable bactericidal and enzyme inactivation effects, and nisin had a significant synergistic lethal effect on aerobic bacteria in orange juice (p < 0.05). Additionally, TSN treatment significantly improved the color attributes of orange juice and well maintained its physicochemical properties and sensory quality. More importantly, TSN treatment significantly increased the total polyphenols content (TPC) and total carotenoids (TC) by 10.03% and 20.10%, increased the ORAC and DPPH by 51.10% and 10.58%, and the contents of total flavonoids and ascorbic acid were largely retained. Correlation analysis of antioxidant activity showed that the ORAC and scavenging ability of DPPH radicals of orange juice are mainly attributed to TC and TPC. These findings indicate that TSN shows great potential application value, which could guarantee the microbiological safety and improve the quality attributes of orange juice.