Paper-based analytics allows building portable and disposable devices for point-of-care (POC) diagnosis. Conventional methods for quantifying proteins exhibit substantial disadvantages related to costs and difficulty of the technique when used in settings where fast and cost-effective assays are needed. We report the successful application of a simple, rapid, easy to use, and label-free aptasensor strategy based on the selective fluorescence of the NMM IX dye. For the probe design, the three-dimensional (3-D) structures of the DNA components were carefully analyzed using software for the 3-D visualization of crystallographic structures. The chimeric aptafluorescence molecule consists of two modules, a detection aptamer and a transduction sequence that induces the specific fluorescence of NMM IX. In the presence of thrombin, a fluorescent spot visible to the naked eye can be observed. The fluorescent response is directly proportional to protein concentration and can be easily quantified colorimetrically using a low-cost microscopy system. The recognition probe design might be adaptable to other relevant biological analytes by changing the sequence of the aptamer. This proof of principle represents a contribution to the development of useful, cheap, reliable, and simple protein quantification assays for POC testing.