Objective To establish a magnetic separation method for rapid and efficient culture of the primary C57BL/6J mouse ophthalmic artery smooth muscle cells (OASMCs). Methods High purity OASMCs were cultured and identified by combining tissue block adhesion method and enzyme digestion method and optimizing the key experimental steps, including collagenase, the formula of agarose iron powder (Ⅱ,Ⅲ) and particle size of iron powder. The specific procedures were as follows: under sterile conditions, the left ventricle of mouse was successively injected with 4 ℃ phosphate buffered saline and agarose iron powder suspension, and the ophthalmic artery was isolated under dissecting microscope. It was cut into tissue blocks of 1.0-1.5 mm, digested with collagenase for 45 min, then separated from the collagenase by magnetic separator, planted evenly in a petri dish, and incubated at 37 ℃ in an atmosphere of 5% CO₂ in air. The OASMCs were subcultured when they reached 85%-90% confluence. The cells from 3 to 7 generations were identified by morphology and immunofluorescence staining. Results The optimal experimental conditions were determined by changing different conditions: the ratio of collagenase to M199 medium was 0.001 5 g∶6 mL, the collagenase digestion time was 45 min, the particle size of iron powder was 5 μm, and the mass ratio of agarose to iron powder was 0.05 g∶0.05 g; the temperature of the agarose iron powder suspension was 37 ℃, and the injector model was 0.6 mm×25 mm TWLB. After 2 d in primary culture, there were long spindle cells crawling out from the tissue blocks of the ophthalmic artery, with a radioactive distribution. After 7 d, iron powder was gradually reduced by replacing new medium, the speed of cell confluence was accelerated, and the cells showed a typical long spindle-shape morphology. Immunofluorescence staining of the cultured OASMCs demonstrated positive α-smooth muscle actin. Conclusion The present method is rapid, efficient, and can achieve high purity cells for culturing primary C57BL/6J mouse OASMCs, which lays a foundation for screening drug activity of ophthalmic artery related diseases.