Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.