Hollow gold nanospheres (HGNs) were generated onto pencil graphite electrode (PGE) by electrodeposition and subsequent displacement reaction between the cobalt nanoparticles and Au3+ ions. HGNs/PGE displayed excellent electrochemical catalytic activities towards dopamine (DA), uric acid (UA) and ascorbic acid (AA). Differential pulse voltammetry was used for the simultaneous determination of DA, UA and AA. The peak separation between UA and DA, DA and AA was 132mV and 157mV, respectively. The lowest detection limits (S/N= 3) were 0.2, 0.7 and 2.5 mM for DA, UA and AA respectively. With good selectivity and sensitivity, the present method has been applied to the determination of DA in injectable medicine and UA in human urine and serum samples