Biosensors fabricated with whole-cell bacteria appear to be suitable for detecting bioavailability and toxicity effects of the chemical(s) of concern, but they are usually reported to have drawbacks like long response times (ranging from hours to days), narrow dynamic range and instability during long term storage. Our aim is to fabricate a sensitive whole-cell oxidative stress biosensor which has improved properties that address the mentioned weaknesses. In this paper, we report a novel high-throughput whole-cell biosensor fabricated by immobilizing roGFP2 expressing Escherichia coli cells in a k-carrageenan matrix, for the detection of oxidative stress challenged by metalloid compounds. The E. coli roGFP2 oxidative stress biosensor shows high sensitivity towards arsenite and selenite, with wide linear range and low detection limit (arsenite: 1.0 × 10(-3)-1.0 × 10(1) mg·L(-1), LOD: 2.0 × 10(-4) mg·L(-1); selenite: 1.0 × 10(-5)-1.0 × 10(2) mg·L(-1), LOD: 5.8 × 10(-6) mg·L(-1)), short response times (0-9 min), high stability and reproducibility. This research is expected to provide a new direction in performing high-throughput environmental toxicity screening with living bacterial cells which is capable of measuring the bioavailability and toxicity of environmental stressors in a friction of a second.