Laser powder bed fusion (LPBF) fabricated Al–Mn–Mg-Sc-Zr alloy generally possesses a bi-modal microstructure of columnar grains and equiaxed grains. Such an anisotropic microstructure would introduce varied tensile performance in different orientations. To date, few study on the microstructure and tensile property anisotropy have been reported for LPBF fabricated Al–Mn–Mg-Sc-Zr alloys built at different layer thicknesses, which limits the exploration in mechanical property optimization. In this work, the microstructure anisotropy and room-temperature tensile properties of LPBF produced and peak-aged Al–Mn–Mg-Sc-Zr alloys built at 30 and 60 μm layer thicknesses were systematically investigated by scanning electron microscope, transmission electron microscope, and tensile testing. A higher layer thickness of 60 μm resulted in coarser grains with the precipitates size remained similarly compared to the 30 μm specimens. This led to a reduced yield strength in 60 μm specimens (492–509 MPa) comparing to those in 30 μm specimens at 502 MPa–510 MPa. In addition, the existence of columnar grains within melt pools contributed to a larger effective slip length in the vertical direction than that in the horizontal orientation. Such difference in effective slip length in the loading direction contributed to a lower strength in vertical orientations. For ductility, the slightly higher defect level in 60 μm specimens (0.58%) resulted in reduced elongations of 3%–6% compared to those of 30 μm specimens (0.10%). The ductility anisotropy was attributed to the preferential distribution of gas pores and keyhole defects at melt pool boundaries.