Objective To establish a micro-Limulus amebocyte lysate (micro-LAL) gel clot test for determining bacterial endotoxin limit of chlorhexidine acetate irrigation. Methods According to the application guidelines of 1143 bacterial endotoxin test and 9251 guidelines for the application of bacterial endotoxin test in the Part Ⅳ of the Chinese pharmacopoeia (2020), a method to eliminate the interference factors of chlorhexidine acetate irrigation was found, and a micro-LAL gel clot test for bacterial endotoxin was established. Results It was confirmed that the interference to the agglutination reaction of Limulus reagent could be eliminated by diluting chlorhexidine acetate irrigation twice with magnesium ion buffer and then diluting it to 8 times with water for bacterial endotoxin test. The bacterial endotoxin limit was 0.5 EU/mL, detected with λ0.06 EU/mL micro-LAL. Conclusion The micro-LAL gel clot test established in this study is of simplicity, accuracy and high repeatability. It is applicable for the examination of bacterial endotoxin limit of chlorhexidine acetate irrigation, and can be used as a quality control method for the biological test of chlorhexidine acetate irrigation.