As a prototypical half-metallic ferromagnet, La0.67Sr0.33MnO3 (LSMO) has been extensively studied due to its versatile physical properties and great potential in spintronic applications. However, the weak perpendicular magnetic anisotropy (PMA) limits the controllability and detection of magnetism in LSMO, thus hindering the realization of oxide-based spintronic devices with low energy consumption and high integration level. Motivated by this challenge, we develop an experimental approach to enhance the PMA of LSMO epitaxial films. By cooperatively introducing 4d Ru doping and a moderate compressive strain, the maximum uniaxial magnetic anisotropy in Ru-doped LSMO can reach 3.0 × 105 J/m3 at 10 K. Furthermore, we find a significant anisotropic magnetoresistance effect in these Ru-doped LSMO films, which is dominated by the strong PMA. Our findings offer an effective pathway to harness and detect the orientations of magnetic moments in LSMO films, thus promoting the feasibility of oxide-based spintronic devices, such as spin valves and magnetic tunnel junctions.