Didodecyl g-biquaternary ammonium salt (DBAS) was synthesized from di-methyl-dodecylamine and epoxy chloropropane by one-step processs. The effects of DBAS dosage, pH value, water temperature and antibacterial time on the sterilization efficiency of heterotrophic bacteria were investigated. The results showed that its sterilization rate was above 90% in 10 h when the dosage was 10 mg/L, pH value from 6 to 9 and water temperature from 25℃ to 45℃. The main anti-bacterial mechanism was that by adsorption, netting and bridging, heterotrophic bacteria were flocculated with DBAS, which made local drug concentration increase to cause the death of bacteria. Both positively electricity quaternary ammonium N+ groups and ?OH made DBAS in water adsorb to the surface of heterotrophic bacteria more easily. Two hydrophobic groups of ?C12H25 and the hydrophilic groups penetrated into the lipid and protein layer inside the membranes of heterotrophic bacteria cells, modified them, and killed heterotrophic bacteria finally.