In this study, the bi-directional fermentation conditions of Ganoderma lucidum and Rosa roxburghii Tratt were optimized by single-factor combined with orthogonal experiments. The content of active ingredients and in vitro antioxidant activity of Ganoderma lucidum fermentation broth and Ganoderma lucidum-Rosa roxburghii Tratt fermentation broth were determined. When the initial pH was 5.50, the Ganoderma lucidum inoculum amount was 4% (V/V) and the addition amount of Rosa roxburghii Tratt was 20 g/L, the polysaccharide content of the fermentation system was high, and the order of influence of the factors was Ganoderma lucidum inoculum amount>addition amount of Rosa roxburghii Tratt>initial pH. Under the optimum conditions of the single factor experiments, compared with the fermentation broth of Ganoderma lucidum alone, the addition of Rosa roxburghii Tratt powder significantly increased the contents of active ingredients such as polysaccharides, flavonoids, polyphenols, and triterpenoids (P<0.05), and theie mass concentrations were 25.20 mg/mL, 41.47 μg/mL, 93.00 μg/mL and 254.55 μg/mL, respectively. The hydroxyl radical scavenging rate, DPPH radical scavenging rate, ABTS radical scavenging rate and reducing power of the Ganoderma lucidum-Rosa roxburghii Tratt fermentation product (3.00 mg/mL) were 51.54%, 84.87%, 94.79% and 0.60, which were significantly higher than those of the fermentation products without Rosa roxburghii Tratt (45.16%, 74.55%, 73.34%, 0.43). With Ganoderma lucidum strain as the fermentation strain and Rosa roxburghii Tratt as the culture medium, the antioxidant properties of the bi-directional fermentation products increased, indicating that the bi-directional liquid fermentation process of Ganoderma lucidum-Rosa roxburghii Tratt had certain advantages over the traditional fermentation process. The finding of this research provides a basis for the development of medicine and food homologous products based on Ganoderma lucidum-Rosa roxburghii Tratt fermentation system.