l-carnitine is a quaternary amine compound essential for eukaryotic metabolism. It is mainly involved in the oxidative decomposition of medium-and long-chain fatty acids and provides energy for the body. Therefore, it is widely used in health care and food additives. As a pivotal transcriptional activator of l-carnitine metabolism, CaiF is notably activated by crotonobetainyl-CoA, a key intermediate product in the carnitine metabolic pathway. Capitalizing on this mechanism, a sophisticated biosensor was ingeniously developed. Nevertheless, it is worth mentioning that the biosensor currently exhibits a relatively restricted detection range, which results in some specific limitations in practical application scenarios. In this paper, we constructed a biosensor based on CaiF and developed a strategy for modifying this biosensor. The structural configuration of CaiF was formulated by computer-aided design, and the DNA binding site was simulated, which was verified by alanine scanning. Functional Diversity-Oriented Volume-Conservative Substitution Strategy of the key sites of CaiF was conducted to extend the dynamic range of the biosensor. The biosensor based on CaiF, which exhibited a considerably expanded concentration response range, from 10 mM-10 mM, was obtained. The response range was 1000-fold wider and the output signal intensity was 3.3-fold higher to that of the control biosensor. These variants may have great value in improving the l-carnitine production process.