Abstract Two new synthetic luminol derivatives with different substituted chains, LC11 (N-(1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)dodecanamide) and TF46 (N-(1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)-13-undecyloxymethyl-3,6,9,12,15-pentaoxahexacosanamide), were mixed with glycolipid GC11 (10-undecyloxymethyl-3,6,9,12-tetraoxatricosyl 2-acetamido-2-deoxy-β- d -glucopyranoside) to form monolayers at the air–water interface. The pure and mixed interfacial Langmuir films were studied by measuring the surface pressure–molecular area isotherms and their morphologies were characterized by Brewster angle microscopy (BAM). The surface pressure–area isotherms showed differences between both luminol compounds. LC11 (single strand acyl chain) displayed a condensed isotherm with a kink point and a high collapse pressure, whereas TF46 (double strand hydrocarbon chains connected to a triethylene glycol (TEG) moiety) displayed a typical liquid-expanded (LE) curve with a low collapse pressure. The two-dimensional compressibility curves confirmed these results. Furthermore, the transition region and the collapse pressure rose up for mixed monolayers with GC11, which may be due to the cushion effect of GC11 in the mixed monolayer. Dot-like domains were observed by BAM for TF46 Langmuir films, contrariwise to some strip-like aggregated domains in the case of LC11. These different morphologies may be attributed to distinct aggregation modes induced by differences in molecular structure. This was further verified by FT-IR spectroscopy of transferred Langmuir–Schaefer films, suggesting that the alkyl chains of TF46 have a more distorted conformation than that of LC11 in the monolayer.