In solid phase bioanalysis, bioagents are incorporated onto solid materials to give them the ability to detect and/or quantify specific target analytes. Previous research has shown that the enzyme horseradish peroxidase (HRP) can be printed on paper without significant inactivation using piezoelectric ink-jet technologies. However, bioanalytical performance of the resulting bioactive papers can be affected not only by the printing conditions and quantity of enzyme deposited, but also by the enzyme spatial distribution and printed enzyme stability. In this study, confocal laser scanning microscopy (CLSM) is used to characterize the inplane and cross-sectional localized enzyme activity of HRP ink-jet printed on fibrous supports with different surface chemistry and composition. Key paper attributes that can affect the analytical performance are identified.