MXenes are 2D nanomaterials that are considered the materials of the future generation due to their high electrical conductivity, good biocompatibility, and ease of functionalization. This research work reports the electrochemical sensing of Vitamin B₆ using the Manganese dioxide-inorganic phosphate/MXene brush-coated Carbon fiber paper electrode (MnO₂-Pi/MXene/CFP) electrode for the first time. The three-dimensional Ti₃C₂T_x MXene nanosheets consisting of highly ordered, vertically aligned nanosheets with electrochemically deposited MnO₂-Pi are capable of yielding a synergistic effect in combination with high electrochemical performance and large surface area of MnO₂-Pi. The reported electrochemical sensor exhibited a wide linear dynamic range (0.06–650 µM) and a low-level detection limit of 0.021 µM. An increase in the anodic peak current confirms the rapid transfer of electrons transfer arising between the Ti₃C₂T_x MXene and MnO₂-Pi. The results attained substantiate that the fabricated sensor has enhanced selectivity, reproducibility, and stability toward the electrochemical determination of Vitamin B₆ in real samples.