Citrus polymethoxyflavones (PMFs) have considerable medicinal, health-promoting, and commercial importance. To provide a stable and reliable source of PMFs, an efficient process of large-scale preparation is warranted. Here, an extraction model for enriching PMFs from citrus fruits was proposed using an enzyme/acid-catalyzed hybrid hydrolysis approach. This method was optimized using response surface methodology (RSM). Furthermore, this model was applied to ten citrus varieties to prepare PMF-rich extracts, and six main PMFs were qualitatively and quantitatively analyzed using UPLC-ESI-MS/MS. Among the ten investigated citrus extracts, nobiletin was the most predominant PMF. The total yields of the six PMFs were ranked as C. unshiu > C. reticulata > C. sinensis, indicating that C. unshiu was the most suitable raw material for PMF preparation. Additionally, the PMF-rich extracts showed beneficial regulatory effects on gut microbiota, highlighting their potential health-promoting and therapeutic functions, which warrant further exploration.