An immunosensor, utilizing immobilized antibody, is a promising sensing device for its high specificity and broad spectrum for detecting object. But physical adsorption is often an obstacle for its luck of enzymatic reactions. Fluorescence anisotropy immunosensor is a method which is, in principle, free from interference of physical adsorption. In this study 3 reagents, FITC, DNS-Cl and PAS, were employed for fluorescent labeling of antibody and lifetime of fluorophore was investigated to achieve optimum sensitivity. PAS, fluorophore with longest lifetime of up to 100 ns, showed the highest sensitivity which is in accordance with the correlation time of rotational relaxation of antibody, suggesting the importance of fluorescence lifetime being comparable with the correlation time of anisotropy decay. Immobilization procedure of antibody was also investigated to reduce interference of physical adsorption. Antibody immobilized on hydrophobic surface showed substantial anisotropy change by adsorption of non-antigenic protein but antibody on hydrophilic substrate showed no detectable anisotropy change. Further improvement of experimental condition will lead to application for microanalysis or implantable sensor. For practical use of this method, fluorescence measurement with higher S/N ratio is still to be attained.