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Di(2-ethylhexyl) phthalate (DEHP) belongs to environmental endocrine disrupting chemicals (EEDCs) and can be rapidly hydrolyzed into the ultimate toxicant mono-2-ethylhexyl phthalate (MEHP). In this study, we used 5-aminofluorescein modified MEHP (MEHP-AF) as a fluorescence tracer to explore the toxicokinetics, including toxicokinetic parameters, absorption and transport across the intestinal mucosal barrier, distribution and pathological changes of organs. While the dose was as lower than 10 mg/kg by intragastric administration, the toxicokinetic parameters obtained by fluorescence microplate method were similar to those with the literatures by chromatography. MEHP-AF can be rapidly absorbed through the intestinal mucosal barrier in rats. In situ organ distribution in mice showed that MEHP-AF was mainly concentrated in the liver, kidney and testis. Our results suggested that the fluorescence tracing technique had the advantages with easy processing, less time-consuming, higher sensitivity for the quantitative determination, In addition, this technology also avoids the interference of exogenous or endogenous DEHP and MEHP in the experimental system. It also can be utilized to the visualization detection of MEHP in situ localization in the absorption organ and the toxic target organ. The results show that this may be a more feasible MEHP toxicological research method. [Display omitted] • MEHP-AF as a fluorescence tracer can be used to explore the toxicokinetics of MEHP. • Toxicokinetic feature got by fluorescence method was similar to this with by HPLC. • The method could be used to detecting visually in situ in the target organs. • The method had virtue with easy handle and high sensitive for quantitative assay. [ABSTRACT FROM AUTHOR]