A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is presented. In this context, emphasis is placed on amperometric biosensors based on LOx that have been immobilized by direct absorption on carbon surfaces, in particular, glassy carbon (GC) and highly ordered pyrolytic graphite (HOPG). The immobilized LOx layers have been characterized using atomic force microscopy (AFM) under liquid conditions and cyclic voltammetry. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). In the presence of lactate with hydroxymethylferrocene (HMF) as a redox mediator in solution, biosensors obtained by direct adsorption of LOx onto GC electrodes exhibited a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. The proposed biosensor also exhibits good operating performance in terms of linearity, detection limit, and lifetime.