A glassy carbon electrode (GCE) was used as a substrate, and cystine (Cys), carboxylated multiwalled carbon nanotubes (MWNTs), and graphene oxide (GO) were ultrasonically mixed. The surface of the GCE was modified, and a Cys-MWNTs-GO-GCE-modified electrode was prepared. Scanning electron microscopy was conducted to characterize the surface morphology of Cys-MWNTs-GO, which had a loose and porous structure. The electrochemical properties of the modified electrode were examined using K3[Fe(CN)6], and the calculated electrode area of Cys-MWNTs-GO-GCE was 7.0397 × 10−6 cm2. Results also showed that the maximum amperometric response of the modified electrode was reached when the pH values of phosphatebuffered saline for vitamin B2 (VB2) and dopamine (DA) were 6.60 and 7.00, respectively. The modified electrode could detect VB2 and DA in the linear concentration ranges of 2.0 × 10−9– 3.0 × 10−4 and 1.3 × 10−8–2.5 × 10−4 mol/L, respectively. Linear equations could be described as follows: Ip(VB2) = 4.6149 + 4.9889c and Ip(DA) = 3.0063 + 1.8508c. The detection limits of VB2 and DA were 1.2 × 10−9 and 1.0 × 10−9 mol/L, respectively (S/N = 3). These results demonstrated the high stability and reproducibility of the detection method with a peak current of RSD = 1.37%. The developed method could be applied to determine the contents of VB2 and DA in urine and blood samples, respectively.