Due to the unique calcium mineral composition of bone, bone-targeting agents that coordinate with calcium or recognize hydroxyapatite (HAp) have been used to deliver drugs or as imaging agents for the therapy and diagnosis of bone-related diseases. To date, various bisphosphonates or acidic oligopeptide-based bone-targeting agents have been explored. However, bisphosphonates can induce osteonecrosis, and acidic oligopeptide-based molecules have not yet been validated in in vivo bone imaging. In this study, we newly designed b-glutamic acid (bE)-based bone-targeting agents by conjugating mono-bE or tri-bE with polyethylene glycol (PEG) labeled with Cy7 dye (termed as bE-PEG-Cy7 or bEEE-PEG-Cy7). In vitro and in vivo studies showed that bEEE-PEG-Cy7 binds more strongly to calciumcontaining particles and HAp than bE-PEG-Cy7, leading to a clearer visualization of bone tissues and knee joints. Compared to alendronate conjugated-PEG-Cy7 (ALN-PEG-Cy7) as a positive control, bEEE-PEG-Cy7 has lower binding affinity to both HAp and bone tissues, whereas it showed less cytotoxicity toward osteoblastic-like cells. Although bEEE-PEG-Cy7 has a lower performance of binding affinity to HAp and in vivo bone tissues, we believe that bEEE-PEG-Cy7 with less toxicity can be used as a bone-targeting agent for in vivo bone tissue imaging.