It is reported that cholesterol (Chol) and TWEEN 80 at a molar ratio of 5:1 can form small unilamellar vesicles (SUVs) using a staggered herringbone micromixer. These phospholipid‐free SUVs (PFSUVs) can be actively loaded with a model drug for targeting hepatocytes via the endogenous apolipoprotein mechanism. PFSUVs particles with compositions of Chol:TWEEN 80 ranging between 1.5:1 and 5:1 (mol/mol) can be produced with a mean diameter of ≈80 nm, but only the high‐Chol formulations (3:1 and 5:1) can retain a transmembrane gradient of ammonium sulfate for active loading of doxorubicin (DOX). Under cryo‐transmission electron microscopy, PFSUVs‐DOX displays a unilamellar bilayer structure with DOX molecules forming spindle‐shape aggregates inside the aqueous core. Relative to PEGylated liposomal doxorubicin (PLD) that exhibits little interaction with cells in various conditions, the cellular uptake of PFSUVs‐DOX is dependent on the presence of serum and enhanced with an increased concentration of apolipoproteins. After intravenous injection, the vast majority of PFSUVs‐DOX accumulates in the liver and DOX is detected in all liver cells (predominantly the hepatocytes), while PLD is captured only by the sinusoidal cells (i.e., macrophages). This report discloses an innovative lipid bilayer vesicle for highly efficient and selective hepatocyte targeting.