Nowadays, fluorescent reactive dyes have limited color shades and brilliance; thus, they are a group of colorants in the textile industry for dyeing of cotton fabric with high demand. This research has designed and synthesized two novel fluorescent reactive dyes based on fluorescein. In this respect, 4,4′-diaminostilbene-2,2′-disulfonic acid and 4-aminophenyl-4-β-hydroxyl ethyl sulfone sulfate ester reacted with the cyanuric chloride, separately. Then those were reacted with fluorescein and obtained two reactive dyes D1 with dual-emission wavelength and D2 with an emission spectrum. The characterizations of two dyes were carried out using the TLC, 1HNMR, 13CNMR, FTIR, elemental analysis, Ultra-violet spectra, and Fluorimeter techniques. The absorption and emission parameters of dyes in solutions were obtained. The result indicated that the parameters of D1 were more than D2. The results showed that two dyes had a positive solvatochromism effect. The synthesized dyes were applied on cotton fabric, and their color characteristics, fixation and exhaustion percentage were studied.