Bruton's tyrosine kinase, an essential mediator of B cell receptor (BCR) signalling, has been validated as an effective therapeutic target in oncology. Development of chemical sensors capable of precise detection of its expression and function is of great importance for cancer diagnosis and therapy. Here, a dual-purpose probe, IB-4, was developed with excellent inhibitory activity (IC50 = 35 nM), and has been demonstrated to be suitable for simultaneous imaging endogenous BTK activity and studying its target engagement in live cells for the first time. The unique turn-on design endows the probe with excellent sensitivity and selectivity toward BTK under both in vitro and in situ settings, and can be further extended to other irreversible inhibitors for protein characterization, quantification and inhibition studies.