This paper investigated the mechanism for the rheological dysfunction of red blood cell (RBC) caused by free radicals. Normal RBCs were collected from healthy volunteers. Fenton system was added to the RBCs and allowed to react for 30 minutes in vitro. The RBC membrane molecules were damaged due to the free radicals reaction. It was found that: (1) The protein configurations changed such as the ratio decreasing in alpha-helix and increasing in both of beta-sheet and coiled-coil; (2) -P=O/-P-O chemical group ratio in phospholipids increases; (3) the molecular rotational correlation times of proteins and lipids were extremely elevated; (4) the membrane shear elastic modulus was significantly enhanced. The present results support that the chemical structure changes led by free radicals are ultimately responsible for the reduction in molecular fluidity of RBC membrane.