Developing new biomolecule-drug conjugates as prodrugs is a promising area for natural products and pharmaceutical chemistry. Herein, a cholesterol-doxorubicin (Chol-DOX) conjugate was synthesized using cholesteryl-4-nitrophenolate as a facile, stable, and controllable activated ester. This approach offers an alternative to the conventional HCl-emitting cholesteryl chloroformate method, semi-empirical theoretical calculations showed that cholesteryl-4-nitrophenolate exhibits moderate reactivity, higher thermodynamic stability, and a lower HOMO-LUMO energy gap compared to cholesteryl chloroformate, suggesting cholesteryl-4-nitrophenolate could be used as a more controllable acylating agent. The structure of synthesized Chol-DOX conjugate was characterized using NMR and MS techniques. Biological properties of the Chol-DOX were analyzed with the comparison of theoretical and experimental data. This work provided a facile and controllable method to synthesize natural lipid-DOX prodrug and offered an in-depth data analysis of the related biological properties.