Secondary reaction of amino acid L-proline with 4-acetamido-o-benzoquinone as a product of the enzymatic oxidation of acetaminophen mediated by mushroom tyrosinase isolated from Agaricus bisporus has been studied by amperometric detection in the batch injection analysis configuration and by ultraviolet-visible spectrophotometry. Actually, this scientific work is focused on finding out whether it would be possible to develop an indirect electrochemical method based on the amperometric biosensing suitable for monitoring of the L-proline in various honey samples. For optimum content of acetaminophen (100 µmol dm–3) and tyrosinase (3.33 µg cm–3), a satisfactory linear range from 0.1 to 0.5 mmol dm–3 L-proline was obtained for absorption maximum at 332 nm. However, it is necessary to mention that the indirect determination of L-proline by the amperometric tyrosinase biosensor still presents a challenge in the field of developing biological sensors for monitoring of this amino acid.