In this paper, the in vitro permeation efficiency of two commonly used transmembrane peptides TAT and R9, the effects on cells and the transmembrane efficiencies of different organs in mice were compared. The nonspecific transmembrane peptides TAT and R9 are short peptides of 11 amino acids and 9 amino acids, respectively, which are able to efficiently enter  cell through  cell membrane. In this study, the fusion proteins TATEGFP and R9EGFP were purified, and the lung cancer cells were treated with the same concentration of active TATEGFP and R9EGFP. The transmembrane activity and efficiency of the two were observed and compared. To explore whether the transmembrane peptide has an effect on the cells, the TATEGFP and R9EGFP active peptides  were injected intraperitoneally into mice to study the target positions of the two in vivo, and the distribution of the effect organs of the two membranes was compared. The results showed that the two fusion proteins TATEGFP and R9EGFP were able to effectively penetrate the membrane, and the two transmembrane peptides had less effect on breast cancer cells. In vivo experiments showed that the two transmembrane peptides were distributed in the heart, liver, spleen, lung and kidney organs. R9 was mainly enriched in the kidney and liver of mice, and TAT was enriched in the six organs studied. The two cellpenetrating peptides can pass through the bloodbrain barrier to reach the brain. Relatively speaking, TAT has a better transmembrane effect in mice and gives more fluorescence enrichment, and R9 overall membrane penetration is weaker. Through the analysis of this study, the cell safety concentration of two transmembrane peptides and the transmembrane ability of living cells were compared, which provided basic data and a guidance for the rational and effective selection and use of TAT and R9 transmembrane peptides in subsequent experiments.