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Special technique was developed for enzymatic activity determination by surface-enhanced Raman spectroscopy. Human cholinesterases have important physiological functions and were chosen as the object of research. The best known method for determining the activity of cholinesterases is to measure substrate and product of the enzymatic reaction. New silver SERS-substrate was discovered, characterized and explored. New developed technique allows determination of BChE concentration and its activity in buffer and blood. Ellman’s assay was used as reference method.