Here, chiral second near‐infrared (NIR‐II) emitting rare‐earth doped silver selenide nanoparticles (R‐ or S‐Ag2Se:Nd/Yd/Er NPs) were fabricated, exhibiting circular dichroism peak at 850 nm and fluorescence peak at 1550 nm, with 145.7‐fold enhanced intensity compared to the reported Ag2Se NPs. Compared with S‐Ag2Se:Nd/Yd/Er NPs, imaging efficiency of R‐Ag2Se:Nd/Yd/Er NPs in living cells was significantly improved due to a higher cellular uptake rate and 927.7‐fold higher affinity. Furthermore, R‐Ag2Se:Nd/Yd/Er NPs reached at the tumor 2‐fold faster than S type of NPs in vivo. We discover that chirality leads to differences in the affinity between chiral Ag2Se:Nd/Yd/Er NPs and cluster of differentiation 44 (CD44) onto the surface of murine mammary carcinoma cell to cause different in vivo imaging efficiency. These results reveal that chiral Ag2Se:Nd/Yd/Er NPs have high photoluminescence intensity and high in vivo imaging efficiency reflecting wide applications in biomedical diagnosis.