AbstractMicroRNA‐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.