Polydiacetylenes (PDAs) have attracted great interested due to their spectacular chromatic transition in response to external stimuli. In particular, application of shear stress induces a colorimetric transition from a non-fluorescent blue phase to a fluorescent red phase. This turn-on fluorescence in response to mechanical stress is sensitive to very small forces, which are typically quite difficult to measure. Described herein is the development of a fluorescent mechanical stress sensor derived from PDAs. First, techniques were developed to fabricate high quality PDA Langmuir films and tune their properties towards different regimes. Using a modified Surface Forces Apparatus (SFA), the fluorescent response of select PDA Langmuir films to mechanical stress was quantified. The PDAs studied here were measured to be sensitive to shear stress on the order of magnitude of kPa. The migration of the slime mold Physarum polycephalum was then examined using PDA Langmuir films. Stresses exerted by the slime mold induced the blue to red transition. The paths explored by the slime mold and a range of stresses were calculated from the fluorescent response. Finally, the structural differences between the blue and red phases PDAs were examined using Grazing Incidence X-ray Diffraction, X-ray Reflectivity and Atomic Force Microscopy.