Differential pH measurement is an electrochemical procedure based on the correlation of variation in pH, measured with a glass electrode, to the quantity of H+ or OH- ions produced by the reaction following the addition of a suitable enzyme. This technique has already been applied in wine analysis coupled with biosensor technology for the determination of L-lactic and L-malic acids. The analysis of total glucose and fructose is based on phosphorylation of the two reducing sugars by ATP and catalyzed by the enzyme hexokinase. This reaction produces H+ ions. An automated software-controlled differential pH meter was used for H+ detection and the pH measured was correlated to the total glucose and fructose concentration present in the sample. Total glucose and fructose was measured in the range 2 to 35 g/L. The procedure based on differential pH-measurement gave results comparable with the reference method of the European Community n. 2676/90 and the classical Fehling method. The percent recovery of glucose and fructose added to wine was 101 to 103 pourcentage, showing excellent accuracy of the differential pH meter analysis. The precision of the procedure was evaluated as satisfactory. The proposed method was simple and rapid because no previous treatment of the sample was required for measurement and data were automatically recorded.