Different strategies, including palladium electrodeposition (PdCV), Pd sputtering (PdS) and Nafion-solubilized carbon nanotube casting (Nafion/CNT), were used to modify screen-printed carbon electrodes (SPCEs) for the fabrication of amperometric enzyme biosensors. The electrochemical properties of the bare and modified SPCEs and the optimal conditions for surface modification were determined. The electrochemical response of the bare SPCE to H2O2 under the potential of 0.3 V could be improved about 100-fold by Pd modification by electrodeposition or sputtering. By contrast, the electrochemical response of the bare SPCE was enhanced by only about 11-fold by Nafion/CNT casting. Moreover, the PdCV-SPCEs exhibited better reproducibility of electrochemical response (a relative standard deviation (R.S.D.) < 6.0%) than freshly prepared PdS-SPCEs (R.S.D. > 10%). The glucose biosensor fabricated from Pd-modified electrodes could be stored for up to 108 days without loosing significant activity. The PdCV-SPCE also showed very reliable signal characteristics upon 50 consecutively repeated measurements of ascorbic acid. The electrocatalytic detection of the Pd-SPCE was combined with additional advantages of resistance to surface fouling and hence good stability. In conclusion, this study demonstrated that deposition of Pd thin film on SPCEs by electrodeposition or sputtering provided superior enhancement of electrochemical properties compared to Nafion/CNT-SPCEs. Despite their high electrochemical response, PdS-SPCEs required an activation process to improve stability and PdCV-SPCEs suffered from poor between electrode reproducibility.