In order to compare the conversion efficiencies of hesperidin into hesperitin-7-O-glucoside by single- and mixed-strain at 30 ℃ and 160 r/min, hesperidin was transformed by submerged fermentation using single and mixed (1:1) cultures of Fusarium nivale and Aspergillus sojae, and the fermentation products and the α-L-rhamnosidase activity of the fermentation broth the at different time points during fermentation were determined by high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and the pH of the fermentation broth was measured as well. The results showed that there was a significant difference between the mixed- and single-strain fermentations in the transformation of hesperidin to hesperitin-7-O-glucoside (P < 0.05). The mixed-strain fermentation gave a higher α-L-rhamnosidase activity (104.81 U/mL) than the single-strain fermentations (24.73 and 26.17 U/mL), and the fermentation of both strains was completed under acidic condition (pH 4-6). Mixed-strain fermentation could provide another way for the comprehensive development of orange peel, which could make full use of citrus by-products and at the same time avoid environmental pollution.