<p>The study presents a method for the immobilization of enzymes on a polydimethylsiloxane membrane probe for membrane inlet mass spectrometry. The method uses an eggshell membrane as a gas-permeable layer containing amino functional groups of lysine residues, which are capable of covalent linkage with enzyme molecules through glutaraldehyde. The membrane probe with immobilized enzymes enables determination of analyte concentrations that do not themselves diffuse through the membrane, provided the analyte participates in an enzymatic reaction that generates or consumes a diffusible substrate or product. This study uses glucose oxidase as a model enzyme and employs it to determine glucose concentrations in complex samples. Wine, fruit juice, milk and bovine blood plasma, as well as possible interferents and substrate analogs, were analyzed using this method and compared with a glucose oxidase amperometric biosensor. The linear response range of the sensor was 50 to 1000 µmol·L^-1 glucose concentration, the response time was approximately 120 s, and the sensitivity was 67.8 µmol·L^-1 decrease in dioxygen concentration per 1 mmol·L^-1 of glucose. The effects of pH, temperature and buffer concentration, as well as stability during long-term storage, were tested. The eggshell membrane with immobilized enzymes was characterized by scanning electron microscopy.</p>