In vivo luminescent imaging in the second biological window (1000–1400 nm, NIR-II) has attracted increasing attention since it can provide high sensitivity to deep tissue in vivo imaging. Herein, we synthesized approximately 10–15 nm-sized NIR-II luminescent nanoparticles (CaF₂:Nd³⁺ NPs). Furthermore, co-doped Y³⁺ was utilized to enhance the NIR-II luminescence of the CaF₂:Nd³⁺ NPs via breaking the aggregation of Nd³⁺. The appearance of a (200) diffraction peak and the broadening of the interplanar spacing of the (111) plane both showed that the incorporated Y³⁺ can dissolve in CaF₂ by occupying the Ca²⁺ sites to form a CaF₂–YF³ solid solution. In particular, the addition of Y³⁺ can greatly enhance the of the NIR-II luminescence of CaF₂:Nd³⁺ NPs. When the Y³⁺ doped concentration reached 0.30, the luminescence intensity of CaF₂:Y³⁺,Nd³⁺ NPs was about 65 times that of CaF₂:Nd³⁺ NPs. In addition, the quantum yield of Ca_0.68Y_0.30Nd_0.02F_2.32 NPs was 9.30% under the excitation of an 808 nm laser with 483 mW cm^？2 power, which was about 3 times higher than that of CaF₂:Nd³⁺ NPs (3.10%). The in vivo imaging results revealed that the in vivo imaging intensity of Ca_0.68Y_0.30Nd_0.02F_2.32 NPs was about 2.38-fold stronger than that of Ca_0.98F_2.02:Nd³⁺_0.02 NPs. All of these results indicated that CaF₂:Y³⁺,Nd³⁺ NPs can be regarded as potential in vivo imaging probes for biological imaging.