Abstract: 【Objective】This study aimed to optimize the optimal medium and inoculation ratio for the co-culture of Bacillus subtilis and Lactobacillus plantarum， and to investigate the interaction mechanism between the two probiotics， providing a theoretical basis and practical guidance for the development of high-efficiency compound probiotic preparations and the optimization of mixed fermentation processes for green aquaculture.【Method】Using Bacillus subtilis 1-5 （BS1-5） and Lactobacillus plantarum 2-7 （LP2-7） isolated from the intestine of Carassius auratus as the research subjects， they were co-cultured in different media at inoculation ratios. The optimal medium and inoculation ratio were determined by monitoring viable cell counts， sporulation rates， and medium pH. Subsequently， contact culture tests were performed to determine the interaction relationship between the two probiotics and the effects of various components of Bacillus subtilis on survival of Lactobacillus plantarum.【Result】All vegetative cells of the BS1-5 died under the environment of pH 3.0， whereas their spores exhibited high survival rates， indicating that spores were more adaptable to acidic conditions than vegetative cells. Both probiotics showed better growth in co-culture liquid medium than in LB or MRS liquid media. The optimal comprehensive growth was achieved when BS1-5 and LP2-7 were co-cultured at a numerical ratio of 5∶1. After 24 h of co-culture， the logarithm values of viable cell counts were 8.58 and 9.13 for BS1-5 and LP2-7， respectively. The sporulation rate of BS1-5 was 91.14%， which was significantly higher than that of single-strain culture and the 10∶1 co-culture at 12 and 24 h （P<0.05， the same below）. When BS1-5 and LP2-7 were cultured in overlapped or spaced patterns on solid medium， no inhibition was observed during their growth. However， the metabolite supernatant of LP2-7 inhibited the growth of BS1-5. After treatments with the cell suspension and broken cells of BS1-5， the logarithm of viable cell counts of LP2-7 was significantly or extremely significantly higher than that of single-strain shake flask culture. The logarithm of viable cell counts of LP2-7 treated with intracellular substances of BS1-5 was also extremely significantly higher than that of single-strain shake flask culture （P<0.01， the same below）， indicating that the vegetative cells， metabolites， and intracellular substances of BS1-5 could mitigate oxygen damage to LP2-7 and promote its growth.【Conclusion】The optimal comprehensive growth effect is achieved when Bacillus subtilis and Lactobacillus plantarum are co-cultured in co-culture liquid medium at a numerical ratio of 5∶1. The acidic environment generated by Lactobacillus plantarum promotes the sporulation of Bacillus subtilis， while the metabolites， vegetative cells， and intracellular substances of Bacillus subtilis mitigate the oxygen damage and enhance the growth of Lactobacillus plantarum. The two probiotics exhibit a mutualistic symbiotic interaction relationship， indicating that a combined microbial formulation of Bacillus and Lactobacillus can potentially be co-fermented and applied in aquaculture.