Tyroserleutide (YSL) is a new anticancer polypeptide developed recently. It can induce hepatocellular carcinoma cells apoptosis and necrosis. However, the subcellular targets of YSL in the hepatocellular carcinoma cells are not known very well. Therefore, a fluorescent bioconjugate of 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRASE)-YSL) was synthesized and purified by native polyacrylamide gel electrophoresis and capillary electrophoresis. After stability was assessed by capillary electrophoresis and fluorescent spectrophotometric assay, concentration of this conjugate was quantitated by fluorescent spectrophotometric assay. Then subcellular distribution of 5(6)-TAMRASE labeled YSL were investigated by laser scanning confocal microscopy to gain a better understanding of the anticancer mechanism of YSL in vitro. The experimental results showed that the 5(6)-TAMRASE can conjugate with YSL stably. When incubated with human hepatocellular carcinoma BEL-7402 cells, 5(6)-TAMRASE labeled YSL could entered the cells in 15 min, and mainly concentrated in the cytoplasm of BEL-7402 cells. Then the fluorescent intensity of 5(6)-TAMRASE labeled YSL which indicated the concentration of them reach the maximum until incubated with the cells for 2 h. After that it began to go down. The 5(6)-TAMRASE could also enter the hepatocellular carcinoma BEL-7402 cells, but it distributed in the whole cell and the intensities had no decrease within 3 h. The results suggest that the distribution pattern of fluorescent labeling YSL is different from that of 5(6)-TAMRASE. The subcellular distribution pattern of 5(6)-TAMRASE labeled YSL was ascribed to YSL and could represent that of YSL. In conclusion, YSL located at the cytoplasm of hepatocellular carcinoma BEL-7402 cells and distributed intensively.