The paper is a review of the researches of Biomolecular Electronics Laboratory concerning the development of biosensors based on electrochemical transducers (amperometric and conductometric electrodes, potentiometric pH-sensitive field effect transistors) and different biorecognition molecules (enzymes, cells, antibodies), biomimics (molecularly imprinted polymers), as sensitive elements for direct analysis of substrates or inhibitory analysis of toxicants. Highly specific, sensitive, simple, fast and cheap detection of different substances renders them as promising tools for needs of health care, environmental control, biotechnology, agriculture and food industries. Diverse biosensor formats for direct determination of different analytes and inhibitory enzyme analysis of a number of toxins have been designed and developed. Improvement of their analytical characteristics may be achieved by using differential mode of measurement, negatively or positively charged additional semipermeable membranes, nanomaterials of different origin, genetically modified enzymes. These approaches have been aimed at increasing the sensitivity, selectivity and stability of the biosensors and extending their dynamic ranges. During the last 25 years more than 50 laboratory prototypes of biosensor systems based on mono- and multibiosensors for direct determination of a variety of metabolites and inhibitory analysis of different toxic substances were created. Some of them were tested in real samples analysis. The advantages and disadvantages of the biosensors developed are discussed. The possibility of their practical application is considere