Polyaniline (PANI) uricase biosensor prepared with template process is reported first in this paper. The fabrication process is as follows. Firstly, a PANI-uricase electrode is obtained using one-step process. Secondly, the electrode is hydrolyzed in 6.0 mol/dm(3) hydrochloric acid solution to remove the uricase that may be affected by aniline monomer from PANI film. Finally, active uricase is immobilized into the PANI film based on the principle of the doping and undoping of the conducting polymer and a PANI-uricase biosensor is obtained. Some factors that affect response current are studied, such as temperature, pH, potential and substrate concentration. The determination of biosensors indicates that the response current of the biosensor prepared by template process decreases only by about 18% for 60 days, but that prepared by two-step process decreases by approximately 39% for 40 h. The uricase in PANI-uricase biosensor prepared by template process mainly interacts with the nitrogen linked to the quinoid ring. The biosensor is characterized with FTIR, UV-Vis and SEM for the first time.