Abstract: Deep sea and islands and reefs in the South China Sea with abundant microbial resources are less affected by human activities than the nearshore waters. As one of the typical coral reef carnivorous fish in the South China Sea, Gnathodentex aurolineatus' intestinal microbiota has a high ability to metabolize carbon sources. To explore cultivable proteinase-producing strains from the intestinal microbiota of typical reef fish in the South China Sea, we selected the proteinase-producing strains from the intestinal microbiota of G. aurolineatus in Zhubi Reef of the South China Sea, and isolated the strain BTZB2. It had a clear hydrolysis circle on the casein plate, and the ratio of hydrolysis circle diameter to colony diameter reached 3.0 at 24^th hour. The strain was identified as Bacillus tropicus on molecular level, combining with morphological and physiological characteristics of bacteria. The optimal conditions for the protease production of this strain were pH of 8.0, salinity of 15‰, temperature of 30 ℃, cultivation time of 48 h and initial bacterial count of 10⁵ CFU·mL⁻¹. After fermentation for 48 h, the protease production activity of this strain reached 319.48 U·mL⁻¹, 156.5% higher than that of the screened strain (124.55 U·mL⁻¹). Under the two optimal conditions: Combination 1 (pH of 7, salinity of 22‰, temperature of 30 ℃, initial inoculation volume of 10⁵ CFU·mL⁻¹) and Combination 2 (pH of 8, salinity of 15‰, temperature of 30 ℃, initial inoculation volume of 10⁵ CFU·mL⁻¹), the specific activities of crude enzyme solution for 24 h were 975.34 and 889.97 U·mg⁻¹, and those for 48 h were 1 836.51 and 2 400.83 U·mg⁻¹, respectively. The results indicate that B. tropicus BTZB2 has a good proteinase production ability, and it can be used as a candidate strain of functional microbial resource from fish in the South China Sea.