The invention belongs to the technical field of microbial strains, and in particular relates to a culture medium for counting live bacteria of Bacillus subtilis, a sterilizing diluent and a method for counting live bacteria. Among them, the solid medium formula: peeled soybean meal powder (crushed to 100 mesh) 4‑6g, corn flour (ground to 100 mesh) 3‑5g, sodium chloride 5‑10g, xanthan gum 3‑5g, agar 11‑ 13g, distilled water 1000mL, pH value 7.3±0.2; sterile diluent: sodium chloride 8‑9g, potassium dihydrogen phosphate 1‑2g, anhydrous glucose 0.3‑0.5g, peptone 0.3‑0.5g, defoamer 0.1 ‑0.2g, 1000mL of distilled water; and the culture method of first adapting to temperature and then cooling down reduces the secretion of antibacterial substances of bacterial cells, reduces the speed of colony expansion, and reduces the competitive inhibition of bacterial cell growth. The method for counting viable Bacillus subtilis of the present invention improves the accuracy of dilution, reduces the death rate of bacterial cells in the oscillation dilution process, reduces the aggregation of bacterial cells, controls the spread of bacterial colonies, reduces the size of bacterial colonies, and improves the efficiency of Bacillus subtilis. Accuracy and repeatability of counting viable bacilli.