Peptidoglycan (PG) is the main substance for cell wall synthesis and plays an essential role in bacterial physiological activities such as proliferation and division. It protects bacterial from external environment and resists pressure generated by bacterial internal expansion. Despite the incredible importance, there is still lack of effective tools for direct imaging and quantitative analysis of PG. Herein, we designed a D-luciferin/luciferase bioluminescent system to visualize and quantify PG levels in living bacterial cells. In our system, we metabolically label PG through aryl azide modified D-amino acid (DAz). Afterwards, TL-PDC, in which the phenolic hydroxyl of D-luciferin is initially protected through “caged” strategy, is introduced to react with DAz through Staudinger ligation reaction and releases the free D-luciferin. This will initiate luciferase catalyzed bioluminescence reaction and generates a strong luminescence signal. It is demonstrated that TL-PDC has good selectivity and sensitivity towards DAz and DAz-labeled PG. Moreover, the system was used to evaluate PG level in living mice by bioluminescent imaging. Most importantly, this method has potential value in biological experiments related to bacterial detection.