Laccase enzyme has been widely used as the catalyst of the biocathodes in enzymatic biofuel cells (BFCs); the poor biocompatibility of this enzyme (e.g., poor catalytic activity in neutral media and low tolerance against chloride ion) and the lack of selectivity for oxygen reduction at the laccase-based biocathode against ascorbic acid, unfortunately, offer a great limitation to future biological applications of laccase-based BFCs. This study demonstrates a facial yet effective solution to these limitations with the assistance of hydrophobic room temperature ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (Bmim(+)PF(6)(-)). With the Bmim(+)PF(6)(-) overcoating, the laccase-based biocathodes possess a good bioelectrocatalytic activity toward O-2 reduction in neutral media and a high tolerance against Cl-. Moreover, the Bmim(+)PF(6)(-) overcoating applied to the laccase-based biocathodes also well suppresses the oxidation of ascorbic acid (AA) at the biocathodes and thereby avoids the AA-induced decrease in the power output of the laccase-based BFCs. The mechanisms underlying the excellent properties of the Binim(+)PF(6)(-) overcoating are proposed based on the intrinsic features of ionic liquid Bmim(+)PF(6)(-). To demonstrate the applications of the BFCs with the as-prepared biocathodes in biologically relevant systems, an AA/O-2 BFC is assembled with single-walled carbon nanotubes (SWNTs) as electrode materials both for accelerating AA oxidation at the bioanode and for promoting direct electron transfer of laccase at the biocathode. With the presence of 0.50 mM AA in 0.10 M quiescent phosphate buffer (pH 7.2), the assembled BFC has an open circuit voltage of 0.73 V and a maximum power output of 24 mu W cm(-2) at 0.40 V under ambient air and room temperature. This study essentially offers a new strategy for the development of enzymatic BFCs with a high biocompatibility.