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In the present study, we introduced cholesterol (CLO)‐conjugated bovine serum albumin nanoparticles (BSA NPs) as a new system for indirect targeting drug delivery. Tamoxifen, as an anticancer drug, was loaded on BSA NPs (BSA‐TAX NPs); CLO was then conjugated to the BSA‐TAX NPs surface for the targeted delivery of NPs system, by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxy succinimide carbodiimide chemistry (CLO‐BSA‐TAX NPs). The physicochemical properties, toxicity, in vitro, and in vivo biocompatibility of the BSA NPs system were characterized on cancer cell lines (4T1). The results revealed that the BSA NPs system has a regular spherical shape and negative zeta‐potential values. The drug release of BSA NPs system has shown controlled and pH‐dependent drug release behavior. BSA NPs system was biocompatible but it was potentially toxic on the cancer cell line. The CLO‐BSA‐TAX NPs exhibited higher toxicity against cancer cell lines than other NPs formulation (BSA NPs and BSA‐TAX NPs). It can be concluded that the CLO, as an indirect targeting agent, enhances the toxicity and specificity of NPs system on cancer cell lines. It could potentially be suitable approaches to targeting the tumors in clinical cancer therapy.

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