This paper examines the general principles of bioluminescent enzymatic toxicity bioassays and describes the applications of these methods and the implementation in commercial biosensors. Bioluminescent Enzyme System Technology (BEST) has been proposed in the bacterial coupled enzyme system, wherein NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BEST was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications. For wide-spread use of BEST, the multi-component reagent ‘Enzymolum’ has been developed, which contains the bacterial luciferase, NADH:FMN-oxidoreductase and their substrates, co-immobilized in starch or gelatin gel. ‘Enzymolum’ can be integrated as a biological module into the portable biodetector-biosensor originally constructed for personal use. Based on the example of ‘Enzymolum’ and the algorithm of creating new enzyme biotests with tailored characteristics, a new approach was demonstrated in biotechnological design and construction. The examples of biotechnological design of various bioluminescent methods for ecological monitoring were provided. The advantages of enzymatic assays are their rapidity (the period of time required does not exceed 3-5 minutes), high sensitivity, simplicity and safety of procedure, possibility of automation of ecological monitoring; required luminometer is easily available
