Abstract Genetically modified bacterial biosensors can detect specific environmental compounds. Here, we attempted to establish a fluorescent microplate method to detect arsenic using recombinant Escherichia coli cells transformed with plasmids harboring three tandem copies of the ars promoter/operator—the gene for green fluorescent protein ( gfp). In the biosensors, one copy of arsR, whose transcription is autoregulated by the ars promoter/operator and ArsR in the genome of E. coli, was placed in trans in another plasmid under the control of isopropyl-1-thio-β- d-galactopyranoside-inducible promoter. First, this manipulation enabled regulation of the arsR expression at an adequate level. Second, the copy number of reporter unit also affected signal and noise. When the plasmid harboring three copies of the reporter unit was used, the signal-to-noise ratio doubled and the detection limit decreased from 20 to 7.5 μg L − 1 As(III), compared to the use of the plasmid harboring one copy of the ars promoter/operator- arsR- gfp. Thus, segregation of arsR from the ars promoter/operator- gfp using two plasmids is effective in regulating the signal-to-noise ratio and the detection limit with the different functions.