A methodology regarding preparation and certification of certified reference material (CRM) of theophylline was presented．The CRM was prepared by repeated recrystallization and drying at 80 ℃ in vacuum condition for 5 h.According to analysis procedure of homogeneity,11 bottles of sample were randomly taken from 500 bottles,in which one bottle was measured for ten times,the other ten bottles were respectively measured for one time,and the results were validated by F test and T test statistical methods.The stability inspection was carried on the short term(one month) and the long term(12 months),and the results indicated that the period for theophylline of storage was 12 months at 20 ℃.The purity of theophylline were determined by two methods eg.mass balance method and differential scanning calorimetry(DSC) method,based on different principles．The qualitative and quantitative analysis of impurities were respectively performed by liquid chromatography ion trap time of flight mass spectrometry(LC-IT TOF) and high performance liquid chromatography with diode array detector(DAD).The content of water in theophylline was measured by Karl-Fischer titrator with drying oven using methanol as solvent.Volatile material in theophylline was determined by temperature gravity analysis(TGA).The determination of losses on drying ash was carried out by the routine method.The uncertainty of mass balance method and DSC methods were respectively evaluated.The result showed that the certified result was 99.80% and expanded uncertainty was 0.14%(k=2).