Gramicidin A, a polypeptide antibiotic forming transmembrane ion channels, has been incorporated into a Langmuir monolayer formed by a semifluorinated alkane (SFA). In this work, partially fluorinated tetracosane, perfluorohexyloctadecane (F6H18), has been applied, aiming at finding a suitable matrix for gramicidin A to be transferred onto solid support for a biosensor design. For this purpose, the physiological conditions were of special interest (mixed monolayers containing low gramicidin proportion and the surface pressure of 30 mN/m). Mixed monolayers of gramicidin and SFA were found to be miscible within the whole range of mole fractions. A very significant increase of the stability of SFA monolayer has been found in the presence of gramicidin, even at such a low proportion as X(gramicidin) = 0.1, which is reflected in a 3.5-fold increase of the collapse pressure value of mixed monolayer as compared to the film from pure SFA. This interesting phenomenon has been interpreted as being due to the existence of a strong dipole-dipole interaction between both film-forming molecules. Opposite sign of the measured electric surface potential for gramicidin and SFA, resulting from different directions of the dipole moment vectors in both film molecules, implies that the ordered, antiparallel orientation of the dipole moments in the mixed gramicidin/SFA system can be responsible for its extremely high stability.