Abstract: Current physical and chemical dosimeters are limited in that they cannot directly measure the biological effects of radiation or detect it within the body. Biosensors based on engineered probiotics demonstrate high stability and safety, can be used to detect ionizing radiation in vivo. In this study, an oral engineered microbial sensor for ionizing radiation detection has been developed. The Escherichia coli Nissle 1917 (EcN) was selected as the chassis strain. Using CRISPR/Cas9 gene-editing technology, the cryptic plasmids of EcN were successfully removed to yield the chassis strain ΔEcN. To design a radiation-responsive gene circuit, the recA promoter from the SOS response pathway was utilized as the radiation response element, while a fluorescent protein served as the reporter element. This system was designed to be induced by mitomycin C and γ-rays. The performance of engineered bacteria with various gene circuits was characterized and optimized, resulting in the selection of improved candidate strains. Animal experiments were approved by the Ethics Committee of Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-521). After comparing the in vivo radiation detection capabilities of these strains, the EC-8, which exhibited higher sensitivity, was identified as the final oral microbial sensor. This research applies synthetic biology principles to design and engineer a probiotic capable of detecting ionizing radiation within the body. The findings offer a novel method for in vivo ionizing radiation detection and lay the foundation for the development of live biotherapeutics for the precise diagnosis of radiation damage.