Objective： To establish and validate a kind of capillary gel electrophoresis（CGE） method which can quantitate the content of plasmid DNA isoforms,and explore its application. Methods： Firstly using the coated capillary,the plasmid DNA（ p16E25） was separated by CGE method. Then the plasmid p16E25 was digested by restriction and nicking endonuclease respectively and separated by CGE to determine which migration peak was supercoiled closed circular（ ccc）,linear or open circular（oc） form. Secondly,CGE method for plasmid DNA isoforms separation was further optimized for fluorescent dyes and loading concentration and then validated for its the specificity,accuracy,linearity,precision,limit of detection（ LOD） and limit of quantitation（ LOQ）. Finally,the method was used to detect the change of plasmid DNA isoforms after treatment for being kept at varied temperature,several freeze-thawed cycles and UV irradiation. Results： After optimization,the content percentage of plasmid DNA isoforms could be well quantitated by CGE method with LIF detector using following parameters, including coated capillary whose total length was 40 cm, the 30.2 cm effective length（ inlet to window）,loading the 4 ng·μL^-1 of plasmid DNA at 4 ℃ with capillary temperature at 20 ℃,and injection at 4 s at 1 378.95 Pa. The established CGE method could accurately quantify the content percentage of three plasmid DNA isoforms（ ccc, linear and oc isoforms） with good resolution. The method had good specificity asthere was no observed interference from substances which were often used as plasmid DNA excipients. Linear range of the method for each of plasmid isoforms was from 0.25 ng·μL^-1 to 8 ng·μL^-1. A good linearity was exhibited between content percentage and loading concentration of each isoform,and the correlation coefficient were 0.997 7,0.999 4 and 0.992 7 for linear,oc and ccc isoforms of plasmid DNA,respectively. Spiking 3%-50% of linear or oc isoform plasmid to the original plasmid,the content percentage of the linear or plasmid exhibited good linearity,and the correlation coefficients were 0.999 9 and 0.997 2,respectively. The linear isoform had good recovery ratio ranged from 104% to 120%,which was better than that of oc isoform,rangingfrom 68% to 94%. The repeatability was determined by detecting three concentrations of high,medium and low concentrations of plasmid DNA （4,2 and 0.5 ng·μL^-1）. The RSD of migration time and content percentage of ccc plasmid were 0.22%-0.54% and 0.38%-2.1%, respectively. The results indicated the method had a good intra-assay repeatability. The intermediate precision was also good. The RSD of migration time was 0.52%-1.1%,and the RSD of content percentage were 3.6% and 0.25% corresponding to the medium and high loading concentration of plasmid DNA,respectively. Precision was better when the concentrations were 2 ng·μL^-1 and 4 ng·μL^-1,respectively. After sensitivity validation,LOQ and LOD of the method were 0.5 and 0.25 ng·μL^-1,respectively. Finally,after full validation,the established CGE method could be used for intended purpose. The method was used to determine the change of plasmid DNA isoforms under different conditions,such as storage temperature,freeze-thawed cycles and UV irradiation. The results showed that the content percentage of ccc plasmid DNA isoform was not affected by stored at -80 ℃ and -20 ℃ for one week. However,after stored at room temperature for one week,the content percentage of oc plasmid DNA increased from 1.39% to 4.16%. Plasmid isoforms alteration was not obviously observed after five freeze-thawed cycles, but the content percentage of oc plasmid DNA was increased from about 1.3% to 1.93% after seven freeze-thawed cycles. When the 1 mg·mL^-1 plasmid was exposed to UV irradiation for 15 min to 3 h,the content percentage of ccc plasmid DNA was markedly decreased from 96.97% to 54.38% along with content percentage of linear and oc plasmid DNA significantly increased. Conclusion： The established capillary gel electrophoresis method is capable of well separating the ccc,linear and oc isoforms of plasmid DNA,with high resolution. The method can be used to accurately quantify the content of different isoforms in a plasmid DNA,and it could detect the change of plasmid DNA isoforms with high sensitivity. Therefore,it will be helpful for control of plasmid DNA purity.