The AND gate functions such that when all inputs are activated the downstream gene will be transcribed and it is off otherwise. To accomplish optimal and targeted gene therapy in solid tumor patients, we have constructed an AND gate genetic circuit and investigated whether it could be activated by low-dose radiation in vitro and in vivo. The enhancement green fluorescent protein (EGFP) expression in different tumor cells transfected with control vector plxsn-EGFP confirmed that 2 Gy of radiation and 1% O(2) for 3 h could activate our AND gate. Besides, the obvious different levels of EGFP expression between 2 and 6 Gy of radiation demonstrated that the AND gate could be regulated by radiation doses. Additionally, through EGFP expression and the codistribution of p53 and HIF-1α in xenografts, we illustrated the targeted activation property of the AND gate and real-time monitoring to hypoxic districts in vivo. Moreover, significant growth inhibition and cell cycle arrest in vitro and apoptosis-inducing effects in vitro and in vivo proved that the AND gate induced ideal antitumor effects. In conclusion, the radiation dose-regulated AND gate genetic circuit could not only effectively monitor the therapeutic process in real-time but also induce ideal antitumor efficacy, and can be further exploited for personal therapy in clinical tumor patients.