The preparation of a glass capillary pattered with lipid layers on which lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH) were regionally adsorbed and its application for simultaneous detection of D-glucose and L-lactate in human serum is described. A lipid layer was formed on the surface of BSA-unabsorbed octadecyltrichlorosilane (OTS) inner wall of a glass capillary. The electrostatic charge of the lipid layer was a key factor for adsorbing the enzymes on the lipid layer. The fluorescence intensities were observed at each enzyme site in the presence of diaphorase (DIA), β-nicotinamide-adenine dinucleotide oxidized (NAD), resazurin, D-glucose and L-lactate. The fluorescence intensities at each enzyme site increased with an increase in the concentration of D-glucose and L-lactate=with the detection limits of 32 μM and 4.9 μM, respectively.