With the severe depletion of coarse flake graphite (a critical raw material) resources, developing and utilizing fine and ultrafine graphite resources have recently attracted attention. Froth flotation is a widely used technique for the initial enrichment of graphite; however, the flotation selectivity decreases significantly along with particle size reduction. Ultrasound pretreatment would be a promising method to improve the flotation of fine particles. As an innovative approach to understand better the flotation response of different flake graphite sizes, this study conducted a comparative analysis based on flotation concentrate yield and ash as well as ash removal rate between the flake graphite with various particle sizes after ultrasound pretreatment. Particle size, X-ray powder diffraction, and scanning electron microscopy and energy dispersive X-ray spectroscopy analyses were used to investigate the effect of ultrasound treatment on mineralogical properties of the flake graphite with varied particle sizes. Process outcomes indicated that the flotation performance of fine flake graphite (mean chord length: 62.63 μm) was significantly enhanced after ultrasound pretreatment. However, flotation of the ultrafine flake graphite (mean chord length: 24.97 μm) after ultrasound treatment was limited due to the difficulty of generating sufficient fragmentation and dissociation by microjets and shock waves formed by the cavitation effect. Compared with conventional flotation, the concentrate yield of ultrasound flotation increased from 88.95 to 94.98%, ash content decreased from 5.72 to 4.87%, and ash removal rate enhanced from 36.94 to 42.61%. Particle size and mineral property analyses confirmed that further crushing and dissociation of the larger flake graphite after ultrasound pretreatment would be the main factors contributing to improved flotation performance. Additionally, the formation of air flocs in the coarse flake graphite during the ultrasound pretreatment process facilitated the flotation recovery of the crushed graphite particles.