A system used for detecting the transcriptional activating activity of the function-unknown gene products in mammalian cells was developed. Based on the plasmid pTet-Off and the eukaryotic expressing vector pCDNA3.1B(-)/myc-his, firstly, we constructed a set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negative control), pZHO2 (as a positive control). The system also includes the plasmids pTRE-luc (encoding the Firefly luciferase reporter gene) and pRL-TK (encoding Renilla luciferase gene as background control). To confirm the feasibility of the system, the plasmids pZHO1, pZHO2 and pZHO3 (encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was contransfected into such mammalian cells as C4-2, MCF-7, COS7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibility of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.