The interaction of protein with lipid membrane as well as their assembly is of immense scientific importance in biomedical areas and others. Here we report the studies on the interaction between DPPC (phospholipid) Langmuir monolayer and the protein hemoglobin (Hb), by Langmuir-Blodgett (LB) technique at different conditions. The DPPC-Hb interaction has been studied by measuring surface pressure (pi)-area (A) isotherms for these monolayers under compression, kinetics of the change in 7 with time in LB trough. The inclusion of Hb into the single layer of DPPC has been found to be dependent on the surface pressure, concentration of Hb and their reaction/interaction time. After transferring the monolayers to solid supports, different scanning probe microscopic (FE-SEM, AFM) results show the formation of circular domains in the nano to micron scale of Hb-DPPC film at the onset of LE to LC phase transition region of DPPC (similar to 7 to 10 mN/m). In addition, spectroscopic data (CD and FTIR) is applied to characterize the secondary structure of the protein. The spectroscopic studies indicate the beta-sheet conformation of Hb within the domain structures in Hb-DPPC complex film with intact heme group. The water molecules is being entrapped within the Hb-DPPC complex domain as indicated by the red shifting as well as larger increment in intensity of the O-H vibrational band. Apart from the contribution of the physical state of the lipid monolayer, some electrostatic interaction is also responsible in the protein-lipid interaction. The overall study may find potential in protein-lipid interaction/self-assembly to design nm to micron scale protein-lipid based novel architecture formation. (C) 2012 Elsevier B.V. All rights reserved.