Abstract: Disclosed are a pharmaceutical formulation characterized in that hydrophobic anticancer drugs are incorporated into a cholanic acid-chitosan complex and a preparation method thereof, and more particularly, the cholanic acid-chitosan complex composing of hydrophobic cholanic acid and hydrophilic chitosan forms self-aggregates in an aquatic environment. The pharmaceutical formulation characterized in that hydrophobic anticancer drugs are incorporated into a cholanic acid-chitosan complex, wherein the cholanic acid-chitosan complex forms self-aggregates, prolongs the drug release period, enhances the selectivity of the complex for tumor tissue, and greatly increases drug loading content when a drug is incorporated into the self-aggregates, compared to chemical bonding, which limits drug incorporation. Thus, the pharmaceutical formulation of the present invention is useful for anticancer chemotherapy.