We showed by random mutagenesis that one-amino-acid substitution at Arg94 in fructosylamino acid oxidase from Ulocladium sp. JS-103 enhanced substrate specificity toward fructosyl valine (FV), a model compound of hemoglobin A(1c) Kinetic analysis showed that the specificity of the R94W mutant enzyme toward FV was 14-fold that of the wild-type enzyme. The mutant enzyme obtained will be useful in developing an, enzymatic measurement method for hemoglobin A(1c)..