Electrochemically active composite film which contains multi-walled carbon nanotubes (MWCNTs) incorporated with poly(brilliant cresyl blue) (PBCB) has been prepared on glassy carbon electrode (GCE) and indium tin oxide (ITO) electrode by potentiodynamic method. The presence of MWCNTs in the composite film (MWCNTs-PBCB) enhances the surface coverage concentration (Γ) of PBCB by 0.25 nmol cm₋₂μg₋₁. The surface morphology of various films deposited on ITO has been studied using scanning electron microscopy and atomic force microscopy, which reveals that MWCNTs incorporated with PBCB. The MWCNTs-PBCB composite film exhibits promising enhanced electrocatalytic activity towards the biochemical compound vitamin B₉ (folic acid). The electrocatalytic response of folic acid at PBCB, MWCNTs and MWCNTs-PBCB composite films have been studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The sensitivity of MWCNTs-PBCB film towards folic acid (177 μA mM₋₁ cm₋₂) is higher than the values obtained for PBCB (0.75 μA mM₋₁ cm₋₂) and MWCNT films (148 μA mM₋₁ cm₋₂). Similarly, the limit of detection of folic acid at MWCNTs-PBCB film (76 μM) is lower than the other two films. Further, the DPV and selectivity studies reveal that the MWCNTs-PBCB film is efficient for folic acid determination in real sample.