4-Iodophenol was applied to an enhancer in the direct detection of horseradish peroxidase (HRP) encapsulated in liposomes by using luminol chemiluminescence (CL). Luminol, 4-iodophenol and hydrogen peroxide permeate into the inner phase of liposomes containing HRP, resulting in the progress of 4-iodophenol-enhanced luminol CL catalyzed by HRP in liposomes. The CL intensity observed in liposomes was a factor of 150 greater than that observed in a lipid-free bulk solution. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 30 compared with that in a lipid-free bulk solution. 4-Iodophenol effectively functioned as an enhancer in HRP-catalyzed luminol CL in liposomes.