The hemoglobin concentration in diluted whole blood has been amperometrically measured using a toluidine blue O covalently modified microcylinder carbon fiber electrode. The modified electrode was prepared by modifying the surface of carbon fiber by cyclic voltammetric oxidation to form a lot of -COOH groups, and then covalently linking toluidine blue O (TBO) by acylation. The surface morphology and stability of the TBO modified electrode are discussed. The electrochemical behaviour of the modified electrode was studied by cyclic voltammetry. The number of H+participating in the surface electrode process and some kinetic parameters in various pH solutions were determined. The TBO modified microelectrode showed significant electrocatalytic activity for the oxidation of hemoglobin(HB) in pH 5.4 solution. The amperometric response to HB increased linearly with increasing concentration from 8.0x10−6to 8.0x10−4M. The obtained HB concentration correlated well with that obtained using a reference procedure.