A method for preparing test samples for visual inspection process of vaccine injection that meets pharmacopoeia requirements was established. First of all, based on the regulatory requirements, product and process evaluation, and the principle of visual inspection equipment, the types and materials of test samples were confirmed. Next, particles corresponding to the types and materials were prepared, and the target particles were screened out by the sieving method, and then added into the product containers. After that, the detection rate was calculated from multiple inspections to evaluate the detectability of particles, thereby selecting suitable defective products. Finally, considering the final use of the test samples, defective products with appropriate detection rates were selected to comprise a test sample set. This method can successfully prepare test samples for the visual inspection process of vaccine injections that meet pharmacopoeia requirements, providing practical and feasible solutions for manufacturers in terms of the type selection, particle size screening and quality evaluation of test samples.