In this study, we have developed an aggregation induced emission (AIE) based simple, sensitive, rapid and convenient biosensor for detection of trypsin and organophosphorous pesticides. The biosensor is composed of tetra-anionic sulphonyl derivative of tetraphenylethylene (Su-TPE) and cationic polyelectrolyte protamine (PrS), assembled by electrostatic interactions without any complex process of integration. The AIE property of Su-TPE/PrS system has been studied, in detail, using different spectroscopic techniques, such as, ground-state absorption spectroscopy, steady-state and time-resolved emission spectroscopy. The AIE phenomenon of Su-TPE molecules induced due to Su-TPE/PrS supramolecular complex formation has been supported by studying Su-TPE/PrS system under various environmental factors. After establishing the AIE phenomenon of Su-TPE/PrS complex, the potential of the sensing platform, for trypsin detection, has been explored based on the principle of trypsin dependent PrS hydrolysis. The detection system is highly sensitive in the linear trypsin concentration range of 0–16 nM with detection limit of 0.22 nM. Furthermore, the sensing platform also shows excellent selectivity towards trypsin. More importantly, based on trypsin inhibition, Su-TPE/PrS platform is also responsive towards organophosphorous pesticide detection. The commendable performance of the Su-TPE/PrS biosensor will enhance future development of rapid detection of trypsin along with fast high throughput screening of organophosphrous pesticides.