As potential materials for photosensitive biosensors, polydiacetylene Langmuir-Blodgett films are of great importance in disease diagnosis. The interactions between tricosa-2,4-diynoic acid (TCDA) and dioctadecyl glyceryl ether-beta-glucosides (DGG) and between 10,12-pentacosadiynoic acid (PCDA) and DGG using surface pressure-area (pi-A) isotherms have been studied in this paper. The analysis of the excess free energies of the isotherms showed that in monolayers TCDA and DGG molecules had good miscibility. In contrast, in PCDA/DGG monolayers molecules repulsed each other in most mixed ratios. The morphologies of pure TCDA, DGG, and PCDA. and mixtures of TCDA/DGG and PCDA/DGG at the air/water interface were investigated using Brewster angle microscopy (BAM). BAM images confirmed the results obtained by pi-A isotherms.