An array-based optical biosensor for the simultaneous analysis of multiple samples in the presence of unrelated multi-analytes was fabricated. Urease and acetylcholinesterase (AChE) were used as model enzymes and were co-entrapped with the sensing probe, FITC-dextran, in the sol-gel matrix to measure pH, urea, acetylcholine (ACh) and heavy metals (enzyme inhibitors). Environmental and biological samples spiked with metal ions were also used to evaluate the application of the array biosensor to real samples. The biosensor exhibited high specificity in identifying multiple analytes. No obvious cross-interference was observed when a 50-spot array biosensor was used for simultaneous analysis of multiple samples in the presence of multiple analytes. The sensing system can determine pH over a dynamic range from 4 to 8.5. The limits of detection (LODs) of 2.5-50 microM with a dynamic range of 2-3 orders of magnitude for urea and ACh measurements were obtained. Moreover, the urease-encapsulated array biosensor was used to detect heavy metals. The analytical ranges of Cd(II), Cu(II), and Hg(II) were between 10 nM and 100 mM. When real samples were spiked with heavy metals, the array biosensor also exhibited potential effectiveness in screening enzyme inhibitors.