Abstract: In the traditional luminescent bacteria toxicity test,the test result is not stable and the use of virulent substance HgCl2 as the toxicity reference substance engenders environmental pollution.In order to solve these problems,the experimental conditions were optimized by a series of experiments and the results indicated that the pH value of the samples should be adjusted to 5.0～9.0 and the best exposure time was 20 min.At the same time,zinc ion was selected as a toxicity reference substance to replace HgCl2.The advantages of using Zn2+ as the reference substance are less environmental pollution,more stable result and lower price,etc.Furthermore,there is a quantitative relationship between the toxicity of Zn2+ and the toxicity of HgCl2.The toxicity ratio of Zn2+ to HgCl2 is about 1/12.5.Using Zn2+ as the toxicity reference substance is a convenient and feasible method to represent the toxicity of both chemicals and complex environmental samples.