An analytical tool to monitor trace phthalate was developed using a microfluidic channel device coupled with a novel electrochemical biosensor. At first, the electrochemical sensor was constructed with biomimetic layers to reveal a large hydrogen over potential by controlling the surface charge and hydrophobicity through assembling with a lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and a cationic molecule (toluidine blue O) bonded to a conductive polymer. The modified electrode possessing a highly negative polarization potential (approximately -1.8 V vs Ag/AgCl) can uptake sparingly soluble phthalate ester (PEs) compounds in aqueous media. Each sensor probe material was characterized employing SEM, AFM, XPS, QCM, TEM, UV-visible, and impedance spectroscopy. The microfluidic channel is used first to concentrate and separate trace amounts of phthalates, and then the sensor probe is installed at the end of channel. Experimental variables affecting the PEs analysis were assessed and optimized in terms of biomimetic layer composition and analytical conditions. The linear dynamic range and detection limits of the PEs were 0.15 nM-10.0 μM and ∼12.5 pM with relative standard deviations <5%. The proposed method was applied to evaluate the effect of endocrine disruptors on mammalian kidney cells, where the cell samples show in-taking percentages between 1.8 and 7.0% to the total PEs according to the incubation time.