The marker-free transgenic Citrus was investigated by combining application of a chemical regulated induction system XVE with, Cre/loxP-mediated site-specific DNA recombination in a single transformation to remove carriers. The system can remove marker gene (nptⅡ) by expression of Cre recombinase system, which was tightly controlled by a system upon induction by -estradiol. The infected internodal stem segments of ‘Succari’ sweet orange as explants were cultured on co-cultivation medium (MS+ 3 mg/L BA), After 3 d, they were transformed onto selection medium (MS+75 mg/L Kan+500 mg/L Cef). After selection, regeneration buds grow, with 1~2 mm explants were cut and transferred onto chemical-inducible medium (MS+ 3 mg/L BA + 500 mg/L Cef+ 2 μmol/L β-estradiol). After 15 d of continuous culture, the express of gfp could be observed successfully by fluorescence microscope. Finally, Molecular detection indicated that the DNA recombination and excision in transgenic Citrus had already occurred after 30 d grafted in vitro. The feasibility that this chemical-inducible excising marker gene system could be applied onto Citrus was proved and therefore, an efficient marker-free transgenic system in Citrus could be established.