The absolute molecular orientation of tolan derivative 4-docosylamino-4'-nitorotolan (DCANT) Langmuir-Blodgett films transferred by a horizontal lifting method was determined by the phase measurement of second-harmonic generation (SHG). The absorption spectra of the multilayers indicated DCANT molecules aligned in an in-plane anisotropy. In the phase measurement of SHG, the absolute molecular orientation of the DCANT multilayer was determined in comparison with the interference pattern of a poled polymethylmethacrylate film doped with p-nitroaniline. whose absolute molecular orientation was known. The results indicated the DCANT molecules aligned in parallel each other and the absolute molecular orientation was perpendicular to the direction of compression at the air/water interface, which was probably achieved during the compression process in a long and narrow trough.