The specific detection of superoxide is challenging because of its higher reactivity and short-lived property. Among the various chemical probes for the detection of superoxide, L-012, a luminol-based chemiluminescent probe, enables the specific detection of extracellular superoxide. Here, we demonstrated that coapplication of the peptide boronic acid proteasome inhibitor, bortezomib, with L-012 significantly increased its luminescence without affecting the background. The increase in the ratio of L-012 luminescence by bortezomib was more than five-fold in both NADPH oxidase-expressing cells and the xanthine oxidase-dependent cell-free superoxide generation system. The application of MLN2238, another peptide boronic acid proteasome inhibitor, also enhanced the luminescence of L-012. In contrast, carfilzomib, an epoxyketone proteasome inhibitor, did not increase luminescence, suggesting that the effects of bortezomib depend on the chemical structure of the peptide boronic acid, but not on its pharmacological effects. The highly sensitive detection of superoxide by the application of bortezomib may become useful in the experimental assessment of oxidative stress and future diagnostic applications, particularly in limited amounts of samples.