We report a nanofluidic device for the label‐free detection of phosphoprotein (PPn) analytes. To achieve this goal, a metal ion chelator, namely 4‐[bis(2‐pyridylmethyl)aminomethyl]aniline (DPA−NH2) compound was synthesized. Single asymmetric nanofluidic channels were fabricated in polyethylene terephthalate (PET) membranes. The chelator (DPA−NH2) molecules are subsequently immobilized on the nanochannel surface, followed by the zinc ion complexation to afford DPA−Zn2+ chelates, which act as ligand moieties for the specific binding of phosphoproteins. The success of the chemical reaction and biomolecular recognition process that occur in a confined geometry can be monitored from the changes in electrical readout of the nanochannel. The nanofluidic sensor has the ability to sensitively and specifically detect lower concentrations (≥1 nM) of phosphoprotein (albumin and α‐casein) in the surrounding environment as evidenced from the significant decrease in ion current flowing through the nanochannels. However, dephosphoproteins such as lysozyme and dephospho‐α‐casein even at higher concentration (>1 μM) could not induce any significant change in the transmembrane ion flux. ... mehrThis observation indicated the sensitivity and specificity of the proposed nanofluidic sensor towards PPn proteins, and has potential for use in differentiating between phosphoproteins and dephosphoproteins.