l-Cysteine (Cys) and reduced glutathione (GSH) are important biothiols which are indicators of diseases risk and health status. However, it is difficult to discriminate them due to their functional similarity. We report an electrochemical oxidation-reduction approach for highly selective detection of Cys over GSH based on synergistic effect of fully fluorinated cobalt phthalocyanine and ordered mesoporous carbon CMK-3 modified on glassy carbon electrode by a drop-removing method. The prepared electrode exhibited five pairs of well-defined redox peaks and presented unexpected electrocatalytic activity not only towards the oxidation of thiols but also towards the reduction of the corresponding oxidation products. Electrochemical discrimination of Cys from GSH was presented relying on their difference in the electrochemical reduction behavior of the corresponding oxidation products. In addition, Cys was detected at a so negative potential range (−0.5–−0.75 V) that the electrochemical interference signals from the coexisting species in biological samples such as ascobic acid, uric acid, and dopamine were avoid. The electrode was successfully applied for the measurements of Cys in real biological samples such as cell lysate, human serum and urine with excellent anti-biofouling ability.