We report the mapping of biocatalytically active surfaces, particularly on an express search for optimal immobilization conditions of the enzyme lactate oxidase by means of scanning electrochemical microscopy (SECM). With this aim, soft stylus SECM probes containing a carbon paste ultramicroelectrode were modified with Prussian Blue yielding reproducible hydrogen peroxide (H2O2).sensors with a sensitivity of 1.6 +/- 0.5 A M-1 cm(-2) for screening applications. The ultramicroelectrode response was stable under harsh conditions of I mM H2O2 during the first hour, while the response decay during the second hour was less than 4% providing sensor suitability for long-term experiments. SECM imaging in contact mode of different lactate oxidase spots containing membranes allowed for a straightforward optimization of the enzyme immobilization conditions on rough screen-printed carbon paste substrates. The resulting lactate biosensor was characterized by improved analytical performance characteristics: a four times enhanced sensitivity (up to 0.3 A M-1 cm(-2)) in comparison to previous reports and a remarkably increased operational stability. (C) 2014 Elsevier B.V. All rights reserved.