Abstract: This study aimed to establish a method for identification of the prescription components of Pule'an Tablet based on DNA barcoding technology. Sixteen samples have been collected from 8 different companies, and their DNA were purified using Plant and Animal Genomic Kits. The amplification rates of ITS2 were both 100%, and the amplification rates of COI were 43.75% and 56.25% for these samples' DNA purified using plant and animal kits, respectively. For ITS2, 12 samples obtained high-quality sequencing traces and then were identified as containing Brassica campestris. The other 4 samples showed crucial SNP peaks in the sequencing traces, which were assigned to be B. campestris and B. nigra on the basis of cloning and sequencing experiments. For the PCR products of 9 samples from COI universal primers, two samples were directly sequenced and identified as aphids, and 7 other samples were subjected to cloning and sequencing experiments. Finally, we obtained 82 clone sequences and found that Apis mellifera was detected only in 5 of the remaining 7 samples, and pathogens or pests were detected in all these 7 samples. To solve the failure of bee source detection caused by exogenous contaminations based on COI universal primers, we designed two new COI primer pairs of Apis genus. The amplification rates of both primer pairs were 43.75%, and they were identified as A. mellifera. A total of 9 bee source-free Pule'an Tablet samples from 3 different batches were produced by the same company, and each batch contained 3 replicates. Thus, we speculated that raw rape pollen materials for these 9 samples was not collected by bees. This study proposes an identification method for the prescription components of Pule'an Tablets based on ITS2 and COI sequences, which will provide scientific basis and technical guidance for quality control and market regulation of Pule'an Tablets.