The accurate detection of tumorous methylglyoxal (MGO) and its detoxifier glyoxalase 1 (GLO1) in living systems is critical for understanding their roles in tumor initiation and progression. To date, the in situ fluorescence detection of endogenous MGO and GLO1 in tumor has not been reported. Herein we developed a near-infrared (NIR) fluorescent probe MEBTD to specifically detect tumorous MGO. Compared with previously reported MGO fluorescent probes, MEBTD exhibits several distinct advantages, including NIR emission, high selectivity with an MGO detection limit of 18 nM, and a 131-fold off-on ratio. The probe could sense GLO1 activity and monitor the therapeutic effect of GLO1 inhibitors by imaging tumorous MGO in a both a real-time and in situ manner, demonstrating that the biological effect of GLO1 inhibitors is dependent on the GLO1 activity. Furthermore, MEBTD enables the visualization of tumorous MGO induced by GLO1 inhibitors in vivo. To the best of our knowledge, MEBTD is the first NIR fluorescent probe for specifically imaging tumorous MGO in living animals, indicating the promising potential for tumor diagnosis and therapeutic evaluation.