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The magnetic properties of Fe-based alloy ribbons are sensitive to stress, and it’s an interesting scientific question whether stress-induced magnetic anisotropy during annealing procedure can be eliminated by tempering. In this paper, the synchrotron radiation technique was used to observe the microstructure of Fe_73.5Cu1Nb₃Si_13.5B₉ amorphous ribbons annealed at 540 ℃ for 30 minutes under 394.7MPa stress and tempered several times at the same temperature. The macroscopic elongation of the samples during stress annealing and tempering was recorded by SupereyesB011 microcamera, and the magnetic anisotropy of the samples was measured by HP4294A impedance analyzer. After fitting the experimental data, it is found that: (a) The lattice anisotropy, macroscopic strain and magnetic anisotropy of the sample show negative exponential attenuation with the tempering times, and their final residual are 19.04%. 98.27% and 31.65%. (b) Multiple tempering can not completely eliminate lattice anisotropy, macroscopic strain and magnetic anisotropy induced by stress annealing. (c) The magnetic anisotropy of the sample has a linear relationship with the lattice anisotropy, but the intercept between the reverse extension line of the relation curve and the longitudinal coordinate is not zero. When the lattice anisotropy is zero, there is still 16.36% magnetic anisotropy. This is different from Ohnuma's conclusion that lattice anisotropy is the direct cause of magnetic anisotropy. (d) The structure anisotropy caused by the residual stress after stress annealing is the main cause of magnetic anisotropy, but it is not the only reason. The directional congregation of agglomerated nanocrystalline grains caused by creep of amorphous substrates during stress annealing is also an important cause of magnetic anisotropy induced by stress annealing. Moreover, the magnetic anisotropy induced by the directional congregation of agglomerated nanocrystalline grains due to the creep of amorphous substrates during stress annealing can not be completely eliminated by tempering.