The tensile plastic deformation behavior of B2-ordered Fe3Al single crystals at room temperature was systematically investigated. The results show that the mechanical properties are strongly orientation dependent. The plastic elongation of crystals with orientation near [110] is as high as 42%. Slip trace anal ysis shows that although slip planes are found to change among {110}, {11 2} and {123} with the change in orientations, the initial slip planes in a ll c ases are {110}. Five-stage work hardening curve including four linear stages and one parabolic stage is obtained; but not all stages are observed in the a ctual deformation of each crystal. In combination with investigations of disloca tion substructure, it is found that deformations in stage Ⅰ～Ⅲ are corresponding to the motion of two-fold superdislocations. The higher work hardening rate of stage Ⅱ is mainly due to the stronger interactions between primary dislocations and secondary dislocations than those in stage Ⅲ. Deformation in stage Ⅳ involved is not only the motion of two-fold superdislocations but also the slip of dissociated superpartials with APB traps and the formation of APB tube, both of which are attributed to the hardening. Deformation in stage Ⅴ is control led by the cross slip of dissociated superpartials. The dominated softening effect of cross slip reduces the hardening rate and leads to the formation of para bolic stage.