Abstract The structure behaviors of Hemoglobin (Hb) are in detail studied by the methods of UV–vis and fluorescence spectra, circular dichroism, negative staining-TEM, FF-TEM and electrochemistry techniques in PEG 6000/Tween 80/Span 80/H2O niosome system. The obtained results show that Hb can be adsorbed and outspread on the surface of the niosome membrane. The intensities of the UV–vis and fluorescence spectra of Hb in Hb/niosome system are greater than those in Hb/H2O system. The structure behaviors of Hb are partially stabilized and protected in Hb/niosome system. With the increase of PEG 6000 content in the niosome system, the content of α-helix structure decreases but the contents of β-sheet and random increase for Hb. The content of β-turn structure is almost independent of PEG 6000 content. The negative zeta potential of Hb and the conductivity of the system all decrease. The zeta potential of the niosome firstly decreases and then increases.