Acute pancreatitis is a serious and potentially fatal disease caused by intracellular trypsinogen activation. Although protease detection has been greatly facilitated by the development of protease probes capable of monitoring protease activation and inhibition, real-time quantitative measurement of protease activity in living cells remains a challenge, and the identification of the cellular compartment for trypsinogen activation is inconclusive. Here we report a novel strategy for developing trypsin sensors by grafting an enzymatic cleavage site into a sensitive location for optical change of chromophore in a single enhanced green fluorescent protein (EGFP). Our designed trypsin sensor exhibits rapid kinetic responses for protease activation and inhibition with a large ratiometric optical signal change. In addition, it has strong specificity, as enzymatic cleavage is not observed with other proteases such as thrombin, cathepsin B, tryptase, and tissue plasminogen activator. Moreover, the developed trypsin sensor allows us for the first time to observe, in real time, trypsinogen activation by caerulein in the pancreatic cancer cell line, MIA PaCa-2 without zymogen granules. These developed protease sensors will facilitate improved understanding of mechanisms and locations of protease activation and further provide screening of protease inhibitors with therapeutic effects.