Biosensing of urea by a biosensor as a direct detection method at ambient temperature and pressure instead of chromatography leads to a significant reduction in processing costs. Amperometric biosensors based on urease immobilization on macroporous polypyrrole (MPPy) and pyrrole on the surface of a Pt electrode were developed. Applying cyclic voltammetry (CV), we demonstrated the synthesis of MPPy using monodispersed polystyrene spheres (460 nm) as a template. CV and chronoamperometric studies were conducted to evaluate the electrochemical current of the modified electrodes. For the electrode with polypyrrole (PPy), the biosensor response was linear in the range of 1.67-8.32 mM (R-2 = 0.99). Sensitivity, detection limit, and response time of this biosensor were 0.0035 mA mM(-1), 2.57 mM, and similar to 7s, respectively. For the electrode with MPPy, the linear range was 0.5-10.82 mM (R-2 = 0.99). For this biosensor, sensitivity, detection limit (S/N = 3), and response time were 0.0432 mA mM (-1), 0.208 mM, and similar to 5s, respectively. The modified biosensor with MPPy showed high stability and desirable selectivity for urea.