A method for testing in-vitro cytotoxicity of mouth cigarette extract comprises the following steps that 1, the mouth cigarette extract is prepared; 2, cell inoculation culture is carried out; 3, the mouth cigarette extract is subjected to contamination; 4, a CCK-8 dyeing experiment is carried out; 5, a result is obtained, and analysis is carried out. The method is characterized in that in-vitro cytotoxicity testing of mouth cigarettes is established for the first time, when testing is carried out, the step of washing cells many times is omitted, and the experiment process is simpler and more convenient. A CCK-8 reagent can be directly added into a cell culture medium, no liquid replacement step is needed, the generated water-soluble formazan product can be directly subjected to light absorption value detection, a dissolving step before detection is omitted, and the experiment period and time are shortened; continuous light absorption value detection can be carried out many times, no additional experiment step needs to be added, and convenience is provided for looking for a proper detecting time point; the toxicity of the CCK-8 reagent on the cells is small, the influence of the background and experiment errors are avoided, and the testing result is good in repeatability and high in stability and sensitivity.