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Laccase adsorption-desorption behaviors on the surface of multi-walled carbon nanotubes (MWCNTs) were investigated using spectrophotometry and voltammetry. The optimum condition for laccase adsorption is 5.0 mg/mL of laccase in 0.01 M phosphate-buffered saline (PBS) at pH 5.0. Laccase adsorption is a reversible phenomenon that is dependent upon the nature of MWCNTs and the concentration of ionic strength in the laccase solution. Chitosan was functionalized as a nanoporous reservoir to minimize laccase desorption. Chitosan was found to protect approximately 97.2% of the adsorbed laccase from MWCNTs during the first six hours of observation. The three-dimensional (3D) biocathode, MWCNTs-laccase-chitosan with a 0.2 cm2 geometric area, was shown to have a stable open circuit potential (OCP) of 0.55 V, a current density of 0.33 mA cm-2 at 0.2 V vs. saturated calomel electrode (SCE), and a stable current for 20 hours of successive measurements. This report provides a new insight into the study of a high-performance laccase-based biocathode via optimization of adsorption and minimization of desorption phenomena.