Femtosecond laser was employed to fabricate nanostructured glassy carbon electrode (GCE) for electrocatalysis applications. Atomic force microscopy image demonstrated the formation of femtosecond laser-induced periodic nanostructure on the surface of GCE. The ratio of the intensities of the D band and G band and the surface oxygen concentration changed insignificantly at the GCE after treatment by femtosecond laser. Enhanced voltammetric currents with reduced peak-to-peak separations were observed for the electrochemical reactions of ascorbic acid (AA), dopamine (DA), uric acid (UA), and acetaminophen (AP) using femtosecond laser-induced nanostructured GCE. The femtosecond laser-induced nanostructured GCE demonstrated similar electrochemical behavior to those claimed for carbon nanotube and graphene modified GCEs in AA, DA, UA, and AP oxidation reactions.