The sol-gel-derived optical array-based biosensor for the simultaneous analysis of multiple analytes as potential markers for Alzheimer’s disease was fabricated. β-Amyloid, acetylcholine and glutamate were selected as the biosensing probes. The fluorescent dye, carboxy SNARF-1-dextran, was co-immobilized with glutamate dehydrogenase and acetylcholinesterase for sensing glutamate and acetylcholine, respectively, while Amplex red and FITC-dextran were immobilized with horseradish peroxidase for determination of β-amyloid and hydrogen peroxide (H2O2). The biosensors exhibited a good performance on simultaneous analysis of multianalytes without obvious cross-interference and the detection limits of H2O2, β-amyloid, glutamate and acetylcholine were 1.57 nM, 0.63 nM, 0.55 μM, and 1.0 μM, respectively. The developed array biosensors were also applied to determine multianalytes in human serum samples spiked with various concentrations of analytes and showed a good analytical performance with dynamic range of 4 orders of magnitude for β-amyloid. Results obtained in this study clearly demonstrate the possibility of using a sol-gel-derived optical array-based biosensor for simultaneous analysis of multiple samples in the presence of important analytes for Alzheimer’s disease.