To investigate the interference factors affecting the spectrophotometric determination of cyclotrimethylene trinitramine (RDX) in wastewater following the oxidation of ozone (O₃), a series of exploring experiments were carried out. With the help of the Rank sum test and F test, the system error between the RDX data determined by spectrophotometry and high performance liquid chromatography (HPLC) was estimated. RDX data in wastewater and standard solution before and after oxidation of O₃ obtained by spectrophotometry and HPLC were compared. The possible interfering substances were detected by liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS). The cause of misludging the spectrophotometric results of RDX was analysed.Results show that the systematic error does not exist between the RDX data determined by two test methods under the given significance level α=0.05. The organic substances in wastewater of RDX are oxidized by O₃ to produce formaldehyde, which interferes with the analysis results, but this part of formaldehyde can be effectively removed by sample pretreatment.At the same time, RDX can not be completely mineralized during the oxidation of O₃, producing low-molecular-weight intermediates, methylenedinitramine, dihydroxy-nitramine and 6-nitro-2, 4, 6-triaza-3, 5-dioxo-hexanal and so on. Among them, the decomposition reaction of methylenedinitramine, dihydroxy-nitramine and other amine-intermediates can occur similar to RDX with concentrated sulfuric acid, formaldehyde formed by the reaction becomes the direct interferencing factors of interfering the determination of RDX by spectrophotometry.