A graphite oxide (GO)/heparin–benzalkonium chloride (C12) composite was synthesized. The composite was characterized by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). XRD data showed that spacing between layers of GO increased from 0.59 to 3.256nm. This enlarged layer spacing suggested that heparin–C12 complex intercalated completely in between layers of GO. FTIR also confirmed intercalation of heparin–C12 complex into GO gallery. In vitro release rate of heparin from GO–heparin–C12 was monitored for 30days. Heparin released at a very fast rate from the composite matrix in the first day. The release slowed down significantly after the first day and continued for 30days. In addition, antibacterial activity of the composite against Escherichia coli (E. coli) and Staphlococcus aureus (S. aureus) was evaluated using zone of inhibition and colony count assays. Both GO–heparin–C12 and GO–C12 clearly showed antibacterial activity against E. coli and S. aureus while GO alone has a relatively low activity against S. aureus and almost no effect on E. coli.