Disperse fluorescent ink was controllably prepared via the cyclic sander grinding process and used for thermal transfer printing on a polyester substrate. Via process optimization, 0.1% pH regulator triethanolamine and 0.15% surfactant NP-12 were added in the disperse fluorescent ink. The lightness L* of the polyester substrate was 101.84 and the saturation C* was 84.21 within thermal transfer printing on the polyester substrate. Compared with flat screen printing, the bending rigidity and bending hysteresis moment of the ink-jet printed polyester substrate presented a slight change. The fluorescent effect of the printed polyester substrate was increased to 165.65, and the fluorescence reflectivity increased 4.1% at the maximum emission wavelength λmax = 510 nm. The color fastness of the printed polyester substrate was improved. In particular, the wet rubbing fastness was enhanced by one grade after the ink-jet thermal transfer printing.