Biological samples such as blood and urine contain a useful information indicating the health status of individuals. Therefore, the measurement of these biomolecules is widely used to diagnose diseases. To develop a new glucagon detection method that does not rely on antibodies, we focused on the glucagon receptor. The glucagon receptor, which is the G protein-coupled receptor (GPCR), is widely expressed in whole tissue. Since the glucagon receptor recognizes glucagon with high specificity and sensitivity, we thought that using its recognition site would be suitable for creating a novel glucagon-sensing probe. In this study, glucagon-binding peptides were screened from a peptide library of about 20 residues selected from the glucagon receptor sequence. We developed a glucagon-sensing probe by modifying the glucagon-binding peptides with a fluorescent dye that increases fluorescence intensity in response to environmental conditions. Further optimizing the amino acid sequence of the probe is expected to further improve the sensitivity and specificity. In the future, the probe is expected to be applied to a diagnostic method for blood glucagon that is more specific and more easily applied than conventional methods using antibodies.