Objective The p27 gene of the simian type-D retrovirus was prokaryotically expressed to establish the flow microsphere immunofluorescence assay for detection of simian type-D retrovirus in nonhuman primates. Methods The gene of p27 was amplified by PCR and linked to the pGEX-4T-1 expression vector digested with the restriction enzymes of EcoR I and Xho I, and the recombinant pGEX-4T-1-p27 plasmid was transfected into the BL21 (DE3) cells for expression of target protein. The form of expressed protein and the optimal time for induction were analyzed by SDS-PAGE. The target protein was purified with GST resin and coupled with magnetic beads to establish the flow microsphere immunofluorescence assay for detection of simian type-D retrovirus in clinical specimens. Results The recombinant protein was expressed in the soluble supernatant, and the optimal time for induction was 4 h. After coupling the protein with the magnetic beads, the flow microsphere immunofluorescence assay was successfully established. Using this method, 81-fold diluted serum could still be detected as positive, and no cross-reaction was found in the positive serum of other pathogens of nonhuman primates, indicating the strong specificity of this method.A total of 24 clinical specimens were tested by this flow microsphere immunofluorescence assay as well as ELISA. Among all the 24 specimens, 3 samples were positive detected by the flow microsphere immunofluorescence assay, of which 2 were positive and 1 was negative by ELISA. The coincidence rate of these two methods was 96%. Conclusions The prokaryotic expression of p27 protein of the simian type-D retrovirus is successful and the flow microsphere immunofluorescence assay with high sensitivity, strong specificity and tiny samples needed is established, which lays the foundation for further application of the multi-flow microsphere immunofluorescence technique.