Abstract: Nano-selenium, as a kind of new functional nanomaterials, has attracted wide attention from all over the word. Compared with inorganic selenium, organic selenium or elemental selenium, nano-selenium has a lot of outstanding features, including high biological activity, low toxicity, and large surface area. Biosynthetic process is regarded as a relatively efficient and environment-friendly pathway to produce nanomaterials. Our previous study found that Bacillus subtilis subspecies stercoris strain XP, as a biocontrol bacteria strain, not only had strong resistance to selenium or salt, but also had strong ability of transform inorganic selenium with higher toxicity into selenium nanoparticle (SeNP) with higher bioactivity and safety. Furthermore, the strain XP has been confirmed to have higher safety and bioactivity compared to those identified strains. But so far, the SeNP yield from selenite reduction by strain XP metabolism is inefficient, which seriously hinders the wide application of this technology. As we all known, fermentation process parameters have great influence on the selenite reduction and SeNP yield. Choosing appropriate parameters can increase in the SeNP production, as well as reduce the cost of SeNP synthesis. Response surface methodology (RSM) has been considered to be an effective method of process parameter optimization. In this study, the synthesis conditions (e.g., selenite concentration, speed of cultivation, dosage of inoculation) were further optimized to improve the biological efficiency of SeNP synthesis by strain XP. The fermentation conditions were optimized by single factor test, Box-Behnken design (BBD), and response surface methodology. Firstly, the effects of different initial Se (IV) concentrations (1-8 mmol/L) in the culture medium, shaker speeds (120-200 r/min), and the amounts of strain XP inoculation (0.5-10%) on SeNP production were tested by single factor experiment. The production of SeNP increased first and then decreased with gradual increase of initial Se (IV) concentrations. With the shaking speed going up from 120 r/min to 200 r/min, SeNP production also increased first and then decreased. When the inoculum amount of strain XP was between 0.5% and 10%, the yield of SeNP enhanced rapidly along with the increase of inoculum amount. The results showed that each selected experiment parameter had a strong influence on the SeNP production within their scopes: 1-4 mmol/L Se(IV), 120-180 r/min, and 2.5%-10%, respectively. Based on it, the optimal range of each indicator was confirmed. Meanwhile, the above three factors were taken as the influencing factors and SeNP production was used as the response index. Secondly, the Box-Behnken response surface methodology (RSM) was applied to optimize the fermentation conditions of strain XP used for SeNP biosynthesis. Finally, the optimal theoretical value of fermentation condition obtained through response surface experiments were verified by actual experiments. The results indicated that the optimal fermentation condition for SeNP biosynthesis by Bacillus subtilis XP were as follows: initial Se (IV) concentration of 3.4 mmol/L, shaker speed of 157 r/min, and inoculum amount of 9.9%. The SeNP production was 1.82 mmol/L under the optimum condition, which was increased by more than 60% over that under normal condition. Moreover, the seed germination experiment was conducted for confirming the bio-activity of SeNP biosynthesis under optimized culture condition. Application of SeNP could effectively stimulate the Indian lettuce seed vigor and promote the germination process.