The valorization of fruit wastes provides a sustainable approach to producing nutrient-rich substrates for microbial applications. Characterizing their compositional and structural properties is essential for evaluating potential use in single-cell protein (SCP) production. This study investigates D-optimal mixture-designed substrates derived from banana peel, mango peel, and avocado seed, focusing on chemical composition, structural features, and functional indices. Proximate analysis revealed high carbohydrate content (~ 82%), moderate reducing sugars (20.6–23.1 g/100 g DM), low moisture (~ 7%), and consistent protein and mineral levels, indicating substrate stability. Structural characterization showed total lignin content of 7.07–8.26%, predominated by acid-insoluble lignin, while soluble sugar profiling confirmed the dominance of fermentable hexoses (glucose: 7.6–9.3, fructose: 6.6–8.1 g/100 g DM). Functional indices, including C/N ratios (27–30) and hexose/pentose ratios (~ 1.9), indicated the metabolic potential of the mixtures, and multi-response optimization suggested an optimal blend (46% banana peel, 22% mango peel, 32% avocado seed; R² > 0.97). These results demonstrate that D-optimal mixture design can produce reproducible, nutrient-balanced substrates with defined structural properties. While the study establishes a foundation for potential SCP applications, actual microbial utilization and fermentation performance remain to be experimentally evaluated.