To develop a method for quantitative determination of roxarsone(ROX) in Oryza sativa, a group of conditions including extractant, leachate, eluant and mobile phase were tested. The results showed that the condition with extractant, 0.05 mol/L NaOH-20 g/L K2HPO4, included the activated solid phase extraction column, with the leachate 1.5 mL 0.05 mol/L CH3COONa-CH3OH(pH=7) and 1.5 mL CH3OH, and with the eluant 13% HCOOH-CH3OH(V/V) solution, and the method of 0.05 mol/L K2HPO4 solution containing 0.3% HCOOH and CH3OH(the volume ratio was 80∶20) as mobile phase was justified an optimal pipeline for the quantitative analysis of high performance liquid chromatography. With the concentration of ROX 0.01-10.00 mg/mL, the linear relationship of the method was optimal(r>0.999). The limits of detection and limits of quantification were 20, 67 ng/kg, respectively. The recoveries of ROX in roots, stems, leaves, hulls and rice of Oryza sativa were 80.0%-87.2%, 74.0%-80.6%、80.0%-83.7%, 81.0%-90.0% and 83.2%-92.7%, and the relative standard deviations were 2.2%-5.9%, 2.4%-4.6%, 2%-2.6%, 3.6%-5.7% and 3.2%-5.5%, respectively.