Up‐conversion (UC) luminescent and porous NaYF₄:Yb³⁺, Er³⁺@SiO₂ nanocomposite fibers are prepared by electrospinning process. The biocompatibility test on L929 fibrolast cells reveals low cytotoxicity of the fibers. The obtained fibers can be used as anti‐cancer drug delivery host carriers for investigation of the drug storage/release properties. Doxorubicin hydrochloride (DOX), a typical anticancer drug, is introduced into NaYF₄:Yb³⁺, Er³⁺@SiO₂ nanocomposite fibers (denoted as DOX‐NaYF₄:Yb³⁺, Er³⁺@SiO₂). The release properties of the drug carrier system are examined and the in vitro cytotoxicity and cell uptake behavior of these NaYF₄:Yb³⁺, Er³⁺@SiO₂ for HeLa cells are evaluated. The release of DOX from NaYF₄:Yb³⁺, Er³⁺@SiO₂ exhibits sustained, pH‐sensitive release patterns and the DOX‐NaYF₄:Yb³⁺, Er³⁺@SiO₂ show similar cytotoxicity as the free DOX on HeLa cells. Confocal microscopy observations show that the composites can be effectively taken up by HeLa cells. Furthermore, the fibers show near‐infrared UC luminescence and are successfully applied in bioimaging of HeLa cells. The results indicate the promise of using NaYF₄:Yb³⁺, Er³⁺@SiO₂ nanocomposite fibers as multi‐functional drug carriers for drug delivery and cell imaging.