Abstract: Using cadmium responsive protein (CadR) as detection element and green fluorescent protein as report element, a cadmium-responsive bacterial biosensor was constructed to detect heavy metal cadmium in water samples. Single factor experiment and orthogonal test for detection condition optimization were applied to improve the relative fluorescence intensity of the biosensor. Finally, the spiked Yangtze River water samples was detected with the constructed cadmium-responsive bacterial biosensor based on the given optimal combination. The results showed that the fluorescence response of the biosensor to cadmium in the concentration range of 0-200 μg/L conformed to the dose-dependent effect. The single-factor test showed that temperature, pH, induction time and cations had significant effects on the sensitivity of cadmium-responsive biosensor, while different anion ligands had no significant effect on the detection results. The orthogonal test showed that the optimal detection conditions for cadmium-responsive bacteria biosensor were as follows: temperature 37 ℃, pH 7, induction time 120 min, and adding Mg²⁺ with final concentration of 50 μg/L. The verifying experiment indicated that under the optimal combination condition, the fluorescence response of the biosensor to the same concentration of cadmium increased by 1.6 times. The detection results of the spiked Yangtze River water samples by the sensors showed their high performance in detecting cadmium in 0-500 μg/L environmental water samples, and the maximum fluorescence output reached 20 times of the background.