The interactions, nature of the organization, and physicochemical properties of alkyltrimethylammonium bromide (C(n)TAB, n = 12, 14, and 16)-gelatin mixed assemblies were investigated by UV-visible spectrometry, surface tensiometry, and fluorimetry techniques. The synergistic interaction between the surfactant and gelatin was established from the decrease in critical micellar concentration (cmc) and the increase in molecular parking area of surfactants with an increase in percentage of gelatin from 0 to 0.4%; for example, the cmc of C-16 TAB decreased from 0.93 mM in water to 0.44 mM in the presence of 0.4% gelatin, whereas its A(min) increased from 134.98 to 325.55 angstrom(2). The fluorescence anisotropy data and polarity parameters of pyrene indicated the progressive change in the anisotropy and micropolarity of the mixed system media with gelatin percentage, respectively. The decrease in aggregation number with an increase in gelatin concentration can be attributed to the enhanced compatibility of surfactants with the bulk microenvironment. The maximum rigidity of the mixed system was also significant from the lifetime data of tyrosine. The formation of Menger micelles on gelatin segments was supported by surface tension and anisotropy data. The overall observations can be attributed to the formation of micelles via gelatin-surfactant aggregates; gelatin segments are localized within the microdomain of these aggregates.