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Conductive biopolymers, an important class of functional materials, have received attention in various fields because of their unique electrical, optical, and physical properties. In this study, the polymerization of heme into hemozoin was carried out in an in vitro system by the newly developed heme polymerase (histidine-rich protein 2 (HRP-II)). The HRP-II was produced by recombinant <i>E. coli</i> BL21 from the <i>Plasmodium falciparum</i> gene. To improve the hemozoin production, the reaction conditions on the polymerization were investigated and the maximum production was achieved after about 790 &#956;M at 34 &#176;C with 200 rpm for 24 h. As a result, the production was improved about two-fold according to the stepwise optimization in an in vitro system. The produced hemozoin was qualitatively analyzed using the Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). Finally, it was confirmed that the enzymatically polymerized hemozoin had similar physical properties to chemically synthesized hemozoin. These results could represent a significant potential for nano-biotechnology applications, and also provide guidance in research related to hemozoin utilization.