Pyranose 2-oxidase (PyOx) from Trametes multicolor, a homotetrameric flavoprotein, catalyzes the oxidation of different aldopyranoses at the C2 to their corresponding 2-ketoaldoses, producing H2O2 as a by-product. To make the enzyme communicate with electrodes for applications in enzymatic biofuel cells and biosensors, redox-active polymeric compounds, called mediators, can be used to accomplish mediated electron transfer (MET). Applications of flexible osmium redox polymers have always been promising for such use in enzymatic biofuel cells and biosensors because they allow the formation of a hydrogel and the formal potential can be varied to suit the application and work as an efficient mediator. In this work, rational and semi-rational protein design, based on the crystal structure and on former studies, was used to improve the enzyme’s characteristics for use in biofuel cells. In homogeneous steady-state characterization, the resulting mutants (T169G/H450G/E542K/V546C, S113E/T169G/H450G/Q461R/V546C and S113E/T169G/ H450G/Q461R/E542K/V546C) showed a significant increase in activity when D-galactose was used as electron donor and either 1,4-benzoquinone or ferricenium ion was used as electron acceptor. The catalytic efficiency increased up to 30-fold. For biosensing applications, these PyOx variants, which showed improved properties for D-galactose as a substrate, were wired with different osmium polymers with formal potentials ranging between -0.140 to 0.270 V. The wild type and the three PyOx mutants and the PyOx were characterized by their pH profile, substrate specificity, and enzyme loading experiments. The kinetic parameters of the biosensing system, maximum reaction rate of the enzyme (Vmax) and Michaelis Menten constant (Km), analytical characterizations for different sugars were also determined.