Objective Through the proficiency validation of testing of mammalian orthoreovirus 3 (Reo3)antibody in laboratory mice, to investigate the capacity of experimental animal quality control laboratories, so as to improve the detection capacity. Methods According to the program approved by CNAS, serum samples after calibration were tested for stability and homogeneity, and numbered randomly. The random samples were issued to the participant laboratories with the Standard Operation Procedure(SOP). The participants must submit the test reports and original records in time. The feedback results were judged by the concordance rate with the anticipated results. Results 27 laboratories from 17 provinces were enrolled in this evaluation project, all of them submitted detection results on time. Results All the 27 laboratories were marked as pass or excellent, with a pass rate of 100%. ELISA was used in 26 laboratories, and immunofluorescence assay was used in one laboratory. Conclusions The ability for detection of Reo3 antibody in laboratory mice in animal test laboratories is high. The implementation of proficiency testing can reflect the inspection level of quality control laboratories.