Direct electron transfer was found to be greatly facilitated for horseradish peroxidase (HRP) in a didodecyldimethylammonium bromide (DDAB biomembrane-like film at a pyrolytic graphite (PG) electrode involving the Fe-III \ Fe-II couple. The heterogeneous electron transfer rate constant k(s), was fitted as 9.0 s(-1) using the non-linear regression analysis of the square wave voltammograms at a series of frequencies and pulse heights. The pH dependence of the formal potential for HRP in DDAB film at medium pH environments suggested one-proton transfer coupled with a one-electron transfer reaction. Scanning electron microscopy (SEM) showed different film morphology for HRP and HRP-DDAB films. UV-vis and reflectance absorption infrared (RAIR) spectra inferred that the heme state of HRP in DDAB film was similar to that in its native state. Circular dichroism (CD) results indicated slight perturbation of DDAB on the second structure of HRP. Thus, the embedded HRP in the biomembrane-like DDAB film showed nearly native structural properties and improved electrochemical characteristics. This has potential value for the basic and applied bioelectrochemistry of enzymes.