This study isolated and identified Bacillus amyloliquefaciens B1 from Carcinoscorpius rotundicauda by carrying out the 16S rRNA sequence analysis, reconstructing the phylogenetic tree based on the Environment for Tree Exploration (ETE3) v3.1.1 belonging to the GenomeNet. By an indirect competitive enzyme-labeled immunoassay, B1 could produce tetrodotoxin (TTX) in MRS was more highly than LB media. After purification, TTX producing ability in B1 could be detected in ELISA assay, high performance liquid chromatography (HPLC). The gel permeation chromatography and gas chromatography were applied to determine the molecular weight of EPS and the concentration of glucose in EPS. The results indicated the highest molecular weight of exopolysaccharides (EPS) estimated 1.33 × 106 Da consisted of glucose (150.09 µg/g). TTX yield was proportional to EPS production in the bacterium. The antimicrobial activities of EPS were determined by agar well diffusion method. Diameter of inhibition zone (mm) of Bacillus amyloliquefaciens EPS on the test microorganisms. The EPS could inhibit against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Vibrio parahaemolyticus ATCC 17802 and Micrococcus luteus ATCC 10240. In silico prediction, TTX might interact with Bacillus amyloliquefaciens via the extracellular domain of noncanonic ABC-type transporter from gram positive bacteria. TTX might also interact with peptidase S54, mistic, metal binding protein of Bacillus subtilis and tryptophan-rich sensory protein of Bacillus cereus. This study provides the understanding of TTX producing Bacillus amyloliquefaciens B1 isolated from Carcinoscorpius rotundicauda.