The invention discloses a vibrio qinghaiensis Q67 based long-term microplate toxicity analyzing method of environmental pollutants. The long-term microplate toxicity analyzing method is established by using the vibrio qinghaiensis Q67 as a tested organism and introducing time factors into the toxicity concentration-effect relationship of the traditional compound on the basis of a microplate toxicity analyzing method. The vibrio qinghaiensis Q67 based long-term microplate toxicity analyzing method of the environmental pollutants is characterized by adopting a specific microplate design and sampling scheme and a microplate culture medium suitable for the growth of the vibrio qinghaiensis Q67, measuring the long-term toxicity effect of the environmental pollutants on the vibrio qinghaiensis Q67 and repeatedly measures the toxicity of the same pollutant through multiple plates to ensure the statistical significance of an experimental result. By measuring the long-term toxicity of a part of the environmental pollutants on the vibrio qinghaiensis Q67, compared with short-term toxicity, the long-term toxicity of the tested environmental pollutants is discovered to be obviously higher than the short-term toxicity of the tested environmental pollutants, wherein the difference of the long-term toxicity and the short-term toxicity of antibiotics is most significant. The invention can actually reflect the toxicity of a site compound having specific action on compounds and more reasonably evaluate the toxicity of the pollutants.