MicroRNA-21　(MiR-21)　has　been　confirmed　to　be　upregulated　in　tumors,　and　its　abnormal　expression　is　closely　associated　with　tumor　occurrence.　However,　the　traditional　imaging　methods　are　limited　to　qualitative　imaging　of　miR-21,　and　no　effective　strategy　has　been　developed　for　monitoring　its　concentration　in　vivo　during　cancer　initiation　and　progression.　Herein,　a　biosensor　is　created　utilizing　a　NIR-II　ratiometric　fluorescent　nanoprobe　to　quantitatively　monitor　dynamic　alterations　in　miR-21　levels　in　vivo.　The　nanoprobe　(termed　DCNP@DNA2@IR806)　is　constructed　by　introducing　IR806　as　a　donor　and　down-conversion　nanoparticles　(DCNP)　as　the　acceptor,　using　DNA　as　linkers.　Upon　miR-21-responsive　initiation　of　the　nanoprobe,　the　1550 nm　fluorescent　signal　of　DCNP　stimulated　by　a　808 nm　laser　(F1550,　808Ex)　increased　because　of　the　close　proximity　of　IR806　to　the　DCNP　and　the　subsequent　non-radiative　energy　transfer　(NRET).　Meanwhile,　the　1550 nm　fluorescent　signal　of　DCNP　stimulated　by　a　980 nm　laser　(F1550,　980Ex)　remained　stable　because　of　the　absence　of　NRET.　This　ratiometric　NIR-II　fluorescent　signal　has　been　confirmed　to　be　a　reliable　indicator　of　miR-21　concentration　in　vivo.　The　strategy　holds　promise　for　further　enhancing　the　understanding　of　microRNAs-based　molecular　mechanisms　underlying　cancer　progression,　laying　a　foundation　for　the　early　diagnosis　of　microRNAs-related　diseases.　©　2024　Wiley-VCH　GmbH.