Hemoglobin (Hb) was immobilized on the chitosan films using glutaraldehyde as a bifunctional agent. Atomic force microscopy (AFM) was used to examine the film surface in order to image the presence of Hb and Fourier transform infrared spectroscopy (FT-IR) was detected to elucidate the structural change of the immobilized Hb. The influences of several immobilization parameters were investigated, the optimum concentration of glutaraldehyde, pH and binding time were determined as 0.7%, 4.5 and 6 h, respectively. The enzymatic assay indicates that the immobilized Hb showed a higher thermal stability than that of free Hb, and the catalytic activity in organic solvents was also enhanced. (c) 2006 Elsevier Ltd. All rights reserved.