A permeability-enhancing flooding system for tight oil reservoirs and preparation and use thereof are disclosed. The permeability-enhancing flooding system is present in a state of oil-in-water droplet and composed of a surfactant, an oil-soluble substance and water, and has an outer phase that is an aqueous solution containing the surfactant, and an inner phase that is the oil-soluble substance; and the surfactant comprises one of nonionic gemini surfactants and anionic gemini surfactants, or a combination of two or more thereof. The preparation process for the permeability-enhancing flooding system is simple to operate, suitable for industrialized production, and low in overall cost, and has obvious application prospects in the field of tight oil reservoirs for an enhanced oil recovery.