Technology has improved human quality of life but it caused several impacts also, due to the various contaminants released in the environment. Among these contaminants, mercury is a major concern because of its high toxicity and ubiquity in the biosphere, being classified as a global pollutant. It can occur in different forms (i.e. soluble, gaseous or solid) and chemical species (e.g. Methylmercury, elemental mercury, Hg (II), etc.), which have very different physico-chemical characteristics that, in turn, determine its cycling and bioavailability. Thus, to assess mercury potential impacts, it is necessary to go beyond the total quantitative determination, developing methods that can measure the toxicity of individual Hg species. In this context, we used a novel technique, a bioluminescent microbial biosensor, which detect only bioavailable mercury species, since bacterial Hg bioavailability is critical to define their risks. Biosensors have large applicabilities in different scientific domains such as environmental biomonitoring, medicine, and food analysis. The chosen biological receptor for the biosensor was the bacteria Escherichia coli MC1061, which is a genetic engineered organism capable of emitting light proportional to amount of Hg that enters its cell. Therefore it is a true mercury bioavailability measurement. In the present study the biosensor was used to detect bioavailable mercury from environmental samples collected at three different locations (open dump, semi-controlled landfill and controlled landfill).The biosensor showed high specificity for Hg (II) and good repeatability. Among the tested samples, collected between September and October 2009, the open dump samples had the highest bioavailable mercury levels compared to other samples from semi-controlled and controlled landfill. Thus, the bioluminescent microbial biosensor technique were sensitive enough to measure bioavailable Hg in landfill samples, and probably in other environmental samples, showing a high potential as an environmental monitoring method.