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Hematocrit (HCT) is expressed as the percentage of red blood cells in the whole blood, it is one of the most highly affecting factors which influences the glucose measurement by using handheld device. In this paper, we present an approach for applying the regularized online sequential learning to hematocrit estimation. The input is the transduced current curve which is produced by the chemical reaction during glucose measurement. The experimental results shown that the proposed approach is promising.