The purpose of this study was to substantiate the possibility of using bioluminescent enzyme test system NADH: FMN oxidoreductase and luciferase for soil contamination analysis. The sensitivity of bioluminescent bioassay was tested on model soils of natural origin: sandy and medium loam soil of sod-podzolic type, heavy loam chernozem. The use of a bioluminescent enzymatic test system to detect soil contamination was carried out on water extracts from soils selected in different districts of Krasnoyarsk and the suburbs. The intensity of luminescence of the enzyme test system NADH: FMN oxidoreductase and luciferase is used as an express analysis for the diagnosis of soil contamination. The integral indicator of pollution was the magnitude of the decrease in the intensity of luminescence, which is due to the inhibition of the enzymatic reaction by pollutants contained in a soil. The absorption properties and pH of water extracts from soils did not effect on the sensitivity of bioluminescent bioassay. The sensitivity of the bioluminescent enzyme test system did not depend on the grain size composition of the sod-podzolic soil. The content of humic matter in the aqueous extract from the chernozem reduced the sensitivity of the enzymatic test system. The effect of humic substances on the inhibition of the enzymatic reaction can be compensated by the variation of NADH in the composition of the reaction mixture. Strong soil contamination was detected in industrial areas of Krasnoyarsk. Soils in the greened urban areas are not contaminated. In the future, bioluminescent enzyme testing can be used as an integral bioassay to diagnose soil contamination when compensating for the interfering effect of humic substances.