A novel flow-through sensor based on electrochemical impedance spectroscopy (EIS) and localized surface plasmon resonance (LSPR) for analyzing biomolecular interactions under flow and static conditions is developed and characterized. The sensor consists of a double-side gold-coated perforated polycarbonate membrane as part of a microfluidic system made of poly(dimethylsiloxane) (PDMS). LSPR and EIS measurements are carried out simultaneously by applying media changes (water to NaCl solutions), unspecific adsorption of bovine serum albumin (BSA), or specific lectin binding on glycopolymer brushes. For BSA binding at the surface, EIS sensor signals mainly contain information from the binding activities at the sensor surface at low frequencies, whereas at high frequencies the change of bulk medium is the main contribution to the EIS signal. Here, the LSPR signal corresponds with EIS signal at high frequency. In contrast, in the case of lectin binding on glycopolymer brushes (3.4 nm thick), where the binding mainly takes place in the brush layer in the vicinity of the surface, LSPR data are correlated with the EIS signals at low frequencies. This leads to the conclusion that the origin of LSPR signals strongly depends on surface coverage and can be specified by simultaneously carrying out EIS measurements.