Seagrass beds are highly productive ecosystems that provide essential habitat and food for various marine species. However, seagrasses have been declining worldwide for decades because of human activities and global climate change. The conservation and restoration of seagrasses has become extremely urgent, especially in China. Recent studies showed that, in the rhizosphere of seagrasses, the organic carbon exudates from plant residues and roots are an important carbon source for Azotobacter. Nitrogen fixation by Azotobacter could provide nitrogen for the growth of the plants, thereby promoting the restoration of seagrasses. Therefore, investigating N2 fixation by Azotobacter in the seagrass rhizosphere is essential since it might play an important role in the nitrogen cycle of the seagrass bed ecosystem. In this study, the growth-promoting activities of the rhizobacteria of eelgrass Zostera marina was investigated by isolating two nitrogen-fixing strains (3A and 4G) from the rhizosphere sediments of Z. marina by using N-free medium. Based on the morphology, physiological and biochemical characteristics, sequence analysis of 16S rDNA, and the nitrogenase structural gene nifH, we identified the 3A bacterium as Thalassospira sp. and 4G bacterium as Bacillus sp.. The optimal culture conditions for the two Rhizobium strains were established, and typical growth curves for the two trains were obtained. In particular, strain 3A was a gram-positive bacterium and had an orange yellow-colored circular colony. In addition, it used D-mannose, D-melezitose, and L-rhamnose as carbon sources. The optimal growth temperature, pH, and salinity of 3A were 31.49℃, 8.18, and 2.585%, respectively. In contrast, strain 4G was gram-negative bacteria and had milky circular colony. It could use a series of hydrocarbons as carbon sources, such as D-cellobiose, D-glucose, D-maltose, sucrose, D-mannitol, and D-trehalose. Its optimal growth temperature, pH, and salinity were 37.27℃, 7.99, and 2.920%, respectively. Previous studies have shown that Thalassospira sp. can promote seagrass growth and increase the content of carotene, soluble sugar, and other substances, indicating that this species is a kind of growth-promoting bacteria. Nitrogen-fixing Bacillus sp. is a typical plant growth-promoting bacterium with strong stress resistance that can produce beneficial plant growth-promoting and antibacterial substances. The nitrogen fixation rates of 3A and 4G were 252.21 nmol C_2H_4/(mL·h) and 196.31 nmol C_2H_4/(mL·h), as revealed by the acetylene reduction method. Comparison with the nitrogen-fixing bacteria reported in other studies revealed that the two strains isolated in this study showed good nitrogen fixation ability. Under suitable growth conditions, the two bacterial strains exhibited logarithmic phase proliferation. Accurate positioning of the bacterial growth logarithmic phase and elucidating the growth characteristics of the bacterial strains could provide basic data for the production of bacterial fertilizers in the future. The bacterial strains obtained in this study might be applied in the future development and application of microbial inoculants and seagrass Azotobacter fertilizers, thereby benefitting the recovery of Z. marina beds in the future.