In this research, screen-printed graphene electrodes (SPGEs) were fabricated on a single-plate electrowetting-on-dielectric (EWOD) digital microfluidic chip. The chip contained T-junction EWOD structures for merging buffer reagent and analyzed droplets and the electrochemical sensor based on SPGEs at the end of T-junction. The EWOD structures consisted of silver EWOD electrodes, a polydimethylsiloxane dielectric layer, and Teflon1AF hydrophobic layers. The electrochemical sensor comprised a working SPGE, a counter SPGE, and a screen-printed silver/silver chloride reference electrode. Both EWOD and electrochemical electrodes were fabricated by a screen-printing technique. The digital microfluidic chip with the SPGE sensor was applied to mix and analyze three biochemical reagents, including potassium hexacyanoferrate ( K-4 Fe(CN)(6)) , nicotinamide adenine dinucleotide (NADH), and hydrogen peroxide ( H2O2) . The electrochemical measurements were conducted using cyclic voltammetry over the concentration ranges of 10-500 mu M , 10-500 mu M , and 1000-7500 mu M for K-4 Fe(CN)(6), NADH, and H2O2 , respectively. The results showed that the detection performances, including sensitivity and detection limit of SPGE sensor on the EWOD chip, were considerably better than those of screen-printed carbon paste electrodes (SCPEs).