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A new genetic approach was developed for increasing specificity of microalgal biosensors. This method is based on the use of two different genotypes jointly to detect a given pollutant: (i) a sensitive genotype to obtain sensitivity; and (ii) a resistant mutant to obtain specificity. The method was tested by the development of a microalgal biosensor for the detection of the explosive 2,4,6-trinitrotoluene (TNT) using a wild-type strain (DcG1wt) of Dictyosphaerium chlorelloides (Chlorophyceae) as the sensitive organism, and a TNT-resistant mutant, obtained from DcG1wt strain by a modified Luria–Delbrück fluctuation analysis. The inhibition of chlorophyll a fluorescence of PSII by TNT was used as the biological signal. Significant differences in maximal fluorescence of light-adapted algae (F_m′) between wild-type DcG1wt cells and TNT-resistant mutants, were observed in all the TNT concentrations tested (from 0.5 to 31 …