Determination of glycerol concentration is an important task of analytical biotechnology. Glycerol is one of metabolites of alcoholic fermentation characterizing taste qualities of drinks and food products. It is used in manufacture of explosives, cosmetic and medicines. Determination of triglycerides and glycerol in human blood is a vital indicator of risk caused by of complications of cardiovascular diseases and systemic disorders of fat metabolism. Enzymatic methods for determination of glycerol are the most accurate and environmentally safe. Taking into account the demand for simple and effective method to determine glycerol in various industries and medicine, the important task is to seek of new strains-producers of glycerol oxidizing enzymes and to develop perspective productive technologies. Early screening of strains – potential producers of glycerol-oxidizing enzymes was performed on selective agar medium. At the initial screening stage 227 fungal strains from genera Aspergillus and Penicillium were tested. Colonies of 27 strains of fungi surrounded by visually detected zones with altered agar pigmentation were sorted out. Aim of this work: to determine localization of glycerol-oxidizing enzymes synthesized by mycelial fungi and carry out electrophoretic analysis. Submerged cultivation of 27 selected strains was carried out. To promote localization of glycerol-oxidizing enzymes extracellular and intra-/cell-bound protein fractions were separated. It was found that the produced enzymes were intracellular or cell-bound. Based on the results of the electrophoretic analysis (fig.) 7 strains of mycelial fungi synthesizing glyceroloxidizing enzymes were selected. It was established that 3 strains (Penicillium sp. 8, A. fischeri MSK F-20394, A. varians 3320) synthesized a protein with molecular weight of approximately ~ 400 kDa, and 4 strains (P. fellutanum, A. carbonarius, A. niger УЗ ПК, Penicillium sp. 1000) – 160–165 kDa. According to the literature reports, glycerol-oxidizing enzymes produced by mycelial fungi may belong to glycerol oxidases (EC 1.1.3.B4, molecular weight ~ 400 kDa), glycerol dehydrogenases (EC 1.1.1.6, molecular weight ~ 160 kDa) and alcohol oxidases (EC 1.1.3.13, mol. weight ~ 160 kDa). Thus, 7 strains of mycelial fungi synthesizing glycerol-oxidizing enzymes were selected for further research.