Novel waterborne fluorinated polyurethane (WFPU) based on hydroxyl-terminated poly(fluoroalkyl methacrylates) (HTPFMA) was prepared successfully. The microstructures and micro-phase separation of WFPU were evaluated through infrared spectroscopy (FT-IR), X-ray diffraction (XRD), dynamic mechanical analysis (DMA), and scanning electron microscope (SEM). FT-IR revealed that the H-bonded carbonyl in hard domains increased with the increasing of fluorine content, resulting in enhancing the extent of micro-phase separation between hard and soft segments. XRD results indicated that the crystallinity of hard segment was enhanced with the increasing of fluorine content. The direct visual microstructures by SEM showed that the micro-phase separation was more significant when the fluorine content increased. DMA also revealed strengthened micro-phase separation with increasing fluorine content. The studies of mechanical properties confirmed that WFPU based on HTPFMA had outstanding mechanical properties. The effects of fluorine content on the static contact angles, surface free energy, and surface composition were also studied. The static contact angles of the WFPU films against water and methylene iodide reached around 108° and 84°, respectively. X-ray photoelectron spectroscopy (XPS) confirmed that the fluorine surface enrichment factor in the outermost 10 nm depth was about 10–18 fold higher than its bulk level.