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Cyclic voltammetry and AC impedance spectroscopy were used for the investigation of properties and permeability of asolectin phospholipid layer and its influence on the voltammetric response of carbon electrodes. Permeability of the layer was tested using two electrochemically active compounds – paraquat as an example of lipophilic pesticide and copper sulphate as an example of hydrophilic substance. The phospholipid layer has suppressed the current response of copper ions effectively even in their large excess to paraquat. By contrast, the current response of lipophilic substance paraquat was almost unaffected by the phospholipid layer. As a result, the slope of calibration straightline for the copper ions was 14‐times lower at the modified electrode than at bare electrode. On the contrary, the slope of calibration curve for paraquat was almost the same at modified electrode compared to bare electrode. Unmodified (CPE) and phospholipid modified (LMCPE) carbon paste electrode were applied to the determination of paraquat by adsorptive stripping differential pulse voltammetry. Detection limits 0.8 and 0.2 µmol/L were obtained from the calibration dependencies for CPE and LMCPE, respectively. SPE was used for preconcentration of paraquat from model water samples and the recovery of the whole procedure was higher than 92%. Practical applications: Improvement in both selectivity and sensitivity to lipophilic pesticide paraquat was observed after modification of the glassy carbon electrode and carbon paste electrode with soybean phospholipids (asolectin). The approach can be used for increasing of sensitivity of analytical methods, for development of electrochemical sensors as well as in research of phospholipid aggregates and its properties. Improvement in both selectivity and sensitivity to lipophilic pesticide paraquat was observed after modification of the glassy carbon electrode and carbon paste electrode with soybean phospholipids (asolectin).