To prepare biologically available Zn-based NCs in aqueous solution, we herein reported the synthesis of aqueous Cu:ZnSe/ZnS NCs with internally doped aqueous Cu:ZnSe NCs as the core template. Due to the dual protection of Cu impurities by the ZnSe core and ZnS shells, the as-prepared Cu:ZnSe/ZnS NCs show excellent stability in the open air, which overcomes the intrinsic instability of traditional aqueous Cu:ZnSe NCs. The as-prepared Cu:ZnSe/ZnS NCs possess extremely good stability, good biocompatibility and lower cytotoxicity, and thus can be used as a promising candidate for fluorescent NC-based biological applications.