All living organisms have evolved and fine-tuned specialized mechanisms to precisely monitor a vast array of different types of molecules. These natural mechanisms can be sourced by researchers to build Biological Sensors (BioS) by combining them with an easily measurable output, such as fluorescence. Because they are genetically encoded, BioS are cheap, fast, sustainable, portable, self-generating and highly sensitive and specific. Therefore, BioS hold the potential to become key enabling tools that stimulate innovation and scientific exploration in various disciplines. However, the main bottleneck in unlocking the full potential of BioS is the fact that there is no standardized, efficient and tunable platform available for the high-throughput construction and characterization of biosensors. Therefore, a modular, Golden Gate-based construction platform, called MoBioS, is introduced in this article. It allows for the fast and easy creation of transcription factor-based biosensor plasmids. As a proof of concept, its potential is demonstrated by creating eight different, functional and standardized biosensors that detect eight diverse molecules of industrial interest. In addition, the platform contains novel built-in features to facilitate fast and efficient biosensor engineering and response curve tuning.