Lysozyme is an enzyme that is responsible for the catalysis of β - 1,4 bond breakage in peptidoglycan residues of Gram-positive bacterium cell walls between N-acetylmuramic acid and N-acetylglucosamine. Lysozyme is commonly applied in food industry and its elevated concentration might indicate a development of leukemia or kidney failure, hence it is necessary to elaborate fast and sensitive tools for its detection.Herein, we present the studies on the elaboration of electrochemical aptasensor for lysozyme determination. For that purpose thiolated aptamer probes were immobilized on gold electrode surface that was subsequently modified with 6-mercapto-1-hexanol as blocking agent. It was found that 2h incubation period is sufficient to form monolayers that efficiently bind to target analyte. Methylene blue at 2 μM concentration was chosen as redox indicator as it provided the highest biosensor response in comparison to other tested electroactive molecules. 5 min. incubation time enabled lysozyme detection within the range from 0.1 fM to 10 nM. The developed biosensor exhibited good selectivity toward target analyte as well as minor response toward a reference DNA aptamer specific for HER2 biomarker. The proposed sensor was also successfully used for discrimination of lysozyme in diluted serum samples.