A facile synthetic route for preparing fluorescent conjugated polymer PPESO3 has been proposed. Compared with a commonly used synthetic procedure, not only is the newly developed procedure simplified but also the dealkylation step with BBr3 is avoided. Therefore, the preparing difficulty and danger are reduced, which is obviously beneficial to the preparation and application of PPESO3 and other PPEs with similar structure. Also, the nonspecific bindings between PPESO3 and proteins were investigated. Encouraged by the results, a rather simple but effective sensor system to assay protease activity was developed. This sensor took advantage of the nonspecific interactions of the ionic conjugated polymers with proteins and the enzyme-catalyzed hydrolysis of proteins. It was known that bovine serum albumin (BSA) could enhance the fluorescence of PPESO3 with an apparent blue shift of the peak emission wavelength. Such phenomena have been successfully utilized to monitor trypsin and pepsin activities in this study for the first time.