Based on self-assembly and electrodepositing, a two-dimensional PMo10V2/GO/PDDA hybrid nanocomposite modified glass carbon electrode (GCE) was successfully fabricated for the first time. Graphene oxide (GO), PDDA, and PMo(10)V(2 )have been modified on GCE surface. Analysis by SEM and FTIR, GO has provided larger specific surface area for improving electrocatalysis activity of PMo10V2. The electrochemical behavior and electrocatalysis of the modified electrode were studied by detailed characterization of cyclic voltammetry (CV) and amperometric (i-t) methods. The experimental results demonstrated that PMo10V2/GO/PDDA/GCE has high stability, fast response, and good electrocatalytic activity towards the oxidation of nitrite ion (NO2-), ascorbic acid (AA), and dopamine (DA) compared to GCE, PDDA/GCE, and GO/PDDA/GCE respectively. Their concentrations are linearly proportional to the amperometric signals. The correlation coefficients of NO2-, AA, and DA are 0.999, 0.998, and 0.998 respectively. The detection limits were estimated to be 0.45 mu M, 0.03 mu M, and 0.18 mu M for NO2-, AA, and DA with the signal to noise ratio of 3. Almost no interference effect was observed for sensing of NO2-, AA, and DA in the presence of each other. The proposed modified electrode was employed for the determination of the NO2-, AA, and DA level in human blood serum with recoveries between 97.35% and 104.2%.