In this study, we synthesized a small molecule fluorescent probe for detecting mono-2-ethylhexyl phthalate (MEHP) named MEHP-AF, which formed by MEHP cross-linked with 5-aminofluorescein (5-AF) through amide bond. MEHP-AF had been purified based on the different physicochemical properties of 5-AF with MEHP. MEHP-AF showed fluorescence characteristics coming from 5-AF and liposoluble property coming from MEHP. After physicochemical characterization, a series of biological studies of its action in cells were carried out. The results indicated that MEHP-AF was a fluorescent probe with strong specificity and high sensitivity. It can visibly track the location of MEHP in HeLa cell or subcellular levels under confocal laser scanning microscopy in situ. This novel fluorescent probe is expected to use for studying its intracellular behavior at the cell level, especially for investigating the interaction between MEHP and cellular molecules.