Abstract An amperometric biosensor was proposed for the enantioanalysis of l-lysine. The biosensor is based on the impregnation of l-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650mV. The biosensor exhibited a linear concentration range between 1 and 100nmol/L with a limit of detection of 4pmol/L. The selectivity of the biosensor is high over other amino acids, such as l-serine, l-leucine, l-aspartic acid, l-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of l-lysine in serum samples and pharmaceutical compounds.