MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled daily at the hospitals, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this paper, we describe a MGMT-activated fluorescence turn-on probe for the rapid no-wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O 6 -benzylguanine and an environment-sensitive fluorophore, SBD. In the presence of MGMT, the enzyme transfers SBD to the protein active site where the hydrophobic surrounding causes the fluorophore to exhibit more than 50-fold fluorescence enhancement. With this probe, bright fluorescence was observed for MGMT-positive, Hela S3 and MCF-7 cells, while MGMT-deficient CHO cells displayed no fluorescence. We believe that this fluorescence activation probe design can also be extended to detect other transferases, for which there are still no effective methods to image them in living cells.