Whole-cell biosensors have attracted considerable interests because they are robust, eco-friendly, and cost-effective. However, most of the biosensors harness the naturally occurring wild-type promoter, which often suffers from high background noise and low sensitivity. In this study, we demonstrate how to design the core elements (i.e., RNA polymerase binding site and transcription factor binding site) of the promoters to obtain a significant gain in the signal-to-noise output ratio of the whole-cell biosensor circuits. As a proof of concept, we modified the arsenite-regulated promoter from Escherichia coli K-12 genome, such that it has a lower background and higher expression. This was achieved by balancing the relationship between the number of ArsR binding sites (ABS) and the activity of the promoter and adjusting the location of the auxiliary ABS. A promoter variant ParsD-ABS-8 was obtained with an induction ratio of 179 (11-fold increase over the wild-type promoter) when induced with 1 μM arsenite. Importantly, the developed biosensor exhibited good dose-response in the range of 0.1 to 4 μM (R2 = 0.9928) of arsenite with a detection limit of ca. 10 nM. These results indicated that the engineered promoter modification approach could be used to improve the performance of whole-cell biosensors, thereby facilitating their practical application.