It is an urgent need to develop new environmentally friendly spinel ferrites with high catalytic efficiency. In this work, a series of Mg-Fe-Al-O spinels with different ratios of Mg/Al were successfully synthesized by the reaction sintering method and were used as a heterogeneous photo-Fenton catalyst for degradation of Rhodamine B (RhB). The effect of different ratios of Mg/Al on the properties of Mg-Fe-Al-O spinel was characterized and analyzed through a range of advanced characterization techniques and DFT calculations. The influence factors on the photo-Fenton reaction catalyzed by Mg-Fe-Al-O spinels were systematically investigated. The results showed that the prepared Mg-Fe-Al-O spinels had larger lattice parameters, wider bandgap, and stronger magnetism, with the Mg content increased. Among them, Mg-9 (Mg0.88Fe1.88Al0.23O4) had the best catalytic performance in the photo-Fenton reaction. The degradation efficiency of RhB reached 98.45%, and the TOC removal efficiency was 83.47%. The elemental valence and PDOS of Mg-9 (Mg0.88Fe1.88Al0.23O4) spinels were closer to MgFe2O4. The photo-generated holes could directly oxidize water and hydroxyl to generate reactive oxygen species ·OH, improving the catalytic activity.