The excellent electrocatalytic activity of a new kind of microstructured carbon material, carbon hollow spheres (CS), toward the oxidation of low-molecular-mass biological thiols including cysteine, homocysteine, and glutathione has been investigated. Comparing with conventional bare glassy carbon (GC) electrodes, an overpotential of 0.40 V is reduced at CS-modified GC electrode with electrooxidation starting at 0.0 V (vs. Ag/AgCl). Based on the electrocatalytic activity of CS, a direct electrochemical method for the determination of low-molecular-mass biothiols is proposed. The proposed method was applied to the determination of biothiols in human plasma and urine with satisfactory results.