An online reactive internal extraction electrospray ionization (iEESI) method was developed for the rapid determination of organic and inorganic speciation information for selenium in poultry tissue samples without complex sample pretreatment. The addition of citric acid as a reducing agent to the internal extraction solvent of methanol/acetic acid (99:1, V/V) for iEESI resulted in the reduction of selenate in the sample to selenite, accompanied by the production of malic acid as an oxidation product. The quantitative analysis of selenate was conducted by using malic acid. By addition of o-phenylenediamine (OPD) to the extractant, inorganic selenium including selenite and selenite reduced from selenate was immediately extracted and converted into 1,3-dihydro-2,1,3-benzoselenadiazole after an in situ reaction. The methanol in the reagent extracted organic selenium compounds from the samples, enabling a rapid quantitative analysis of organic and inorganic selenium in chicken tissue. The concentrations of selenomethionine (SeMet), l-selenocystine (SeCys(2)), selenite, and selenate in different tissue samples were examined, showing strong linearity in the range of 1.00-100.00 μg/kg with the limit of detection (LOD) ranging from 0.36 to 0.84 μg/kg. The results were validated by two conventional methods with an accuracy of 84.2-107.6%. The quantitative analysis of three chicken visceral samples revealed that the highest concentration of organic selenium was present in chicken livers, while the highest concentration of inorganic selenium was observed in gizzards. The findings may provide a reliable method for studying selenium metabolism pathways in organisms.