The influences of different pre-tensile strain amounts (8%, 20%, 30% and 40%) on the work hardening behavior of 304L austenitic stainless steel was systematically investigated by scanning electron microscopy (SEM), electron backscatter diffraction (EBSD) and tensile testing. The results indicate that as the strain increases, the effective average grain size of test steel decreases from 5.12 μm to 3.85 μm, and the aspect ratio of grain gradually increases to 2.8∶1. Strain-induced martensitic transformation occurred during deformation,with the volume fraction of deformation martensitic increasing from 0.47% to 3.2%. The tensile strength of test steel increases from 638.06 MPa to 920.51 MPa, the yield strength increases from 320.52 MPa to 897.67 MPa. The work hardening ability of test steel increases with the increasing of strain, its main strengthening mechanisms are twinning strengthening, deformation martensite strengthening and dislocation strengthening. When the strain is 0%-20%, twinning strengthening is dominant, and the test steel has good plastic elongation and strength.