Biosensors can be a powerful tool for real-time monitoring of specific small molecules and for precise control of gene expression in biological systems. Thus, biosensors have attracted much attention for monitoring increasing number of molecules. However, strategies to tune the properties of biosensors remain less explored, which might restrict their wide applicability. Here we report the development of tunable biosensors for monitoring putrescine, an important member of biological polyamines, in Escherichia coli. The native putrescine-responsive PuuR repressor protein was employed as a sensing component, and its cognate operator was installed in engineered promoters to control the expression of downstream green fluorescent protein (GFP) mut3 as a reporter protein. The engineered biosensors were specific for putrescine, and the response time could be modulated by altering growth medium of the biosensor strains. In addition, the response dynamics and detection ranges of the biosensors can be tuned at the genetic level by modulation of PuuR expression, and by manipulation of the chromosomal genes involved in putrescine biosynthesis. To demonstrate utility of the biosensors, we were able to monitor the changes of endogenous putrescine levels caused by genetic manipulations. Furthermore, a link between the excretory putrescine titer and intracellular GFP fluorescence was established for an E. coli strain that was engineered for improved putrescine biosynthesis and excretion. This study provides a strategy for engineering synthetic biosensor circuit for monitoring and tuning the dynamics in sensing putrescine, which can be generally applicable for monitoring other chemicals through taking a similar approach in circuit design.