Due to the fundamental roles sensors play in the study of biological, chemical and environmental processes, considerable efforts have been directed towards the synthesis of robust systems capable of detecting a wide range of analytes with high selectivity and sensitivity over a range of analyte concentrations. This thesis presents an investigation into the design and synthesis of novel 'click' generated chemo- and bio-sensors and molecular machines for fluorescence sensing applications in vitro and in vivo. The introductory chapter highlights recent and relevant examples of 'click'-derived chemosensors that use charge and energy transfer processes as transduction mechanisms for analyte detection. The synthesis and application of fluorescence- based Zn(II) sensors prepared using 'click' chemistry is then described. These sensors have proven to be effective tools for the selective detection of Zn(II) in vitro and in vivo and are synthetically simple to prepare using modular routes. The third chapter describes the synthesis of a series of novel fluorescent [2]rotaxanes which display cation sensing properties, with extremely varied photo-physical properties resulting from small structural changes. A novel approach to bio-sensing is then introduced, based on an 'allosteric scorpionate' model. In these systems, electron paramagnetic resonance (EPR) spectroscopy is used to detect the remote interaction between 'click'-generated Cu(II)-azamacrocyclic complexes containing biotinylated pendant arms and their biological target.