Azide is an important chemical functional group and has been widely used in chemical biology. However, the impact of azide on the in vivo behaviors of compounds has been rarely studied. Herein, azide was introduced into a fluorescent dye for the near-infrared window two (NIR-II) bone imaging. Specifically, we designed and synthesized the small-molecule NIR-II dyes, N3-FEP-4T capped with azide and FEP-4T without azide capping. In vitro assays revealed that N3-FEP-4T showed 5- and 5.6- times higher hydroxyapatite accumulation and macrophage uptake than those of FEP-4T, respectively. Moreover, N3-FEP-4T displayed higher bone uptakes and much better bone NIR-II imaging quality, demonstrating the specific bone-targeting ability of the azide-containing probe. N3-FEP-4T was then further successfully used for osteoporosis NIR-II imaging. Overall, our study provides insights into the impact of azide on the in vivo behavior of azide-containing compounds and opens a new window for biological application of azide.