Endo-β-N-acetylglucosaminidases (ENGases) are used in glycoprotein analysis and glycan remodeling. However, existing ENGase activity assays are complex and unsuitable for high-throughput analysis. This study describes the development of FRET-based glycan probes for simple and real-time detection of ENGase activity. We designed novel probes, containing a fluorophore and a quencher flanking the chitobiose moiety, utilizing Förster resonance energy transfer (FRET) quenching. We synthesized di-, tri-, and pentasaccharide probes and evaluated their quenching efficiencies and suitability for activity detection. The pentasaccharide probe, MM3D, was efficiently cleaved by Endo-M, resulting in increased fluorescence. We then tested the MM3D probe with various ENGases (Endo-H, Endo-F3, Endo-Om, Endo-CC, Endo-S, and Endo-D). While Endo-M, Endo-CC, and Endo-Om activities were detected, Endo-H activity was not, suggesting that ENGase substrate specificity varies depending on the enzyme’s origin. These results demonstrate that the developed FRET-based glycan probes enable simple and real-time detection of ENGase activity. Further, we synthesized a library of probes with diverse glycan structures. Using this library, we evaluated the activities of six commercially available ENGases and observed their distinct substrate specificities. This probe library offers a valuable tool for the simple detection of ENGase activity and will contribute significantly to advances in glycobiology research.