Objective To establish an immunofluorescence assay ( IFA) method of detecting the mammalian orthoreovirus 3 ( Reo3) in animal-derived biological materials and biological products for human use． Methods Reo3-sensitive cells were screened． The virus TCID50 was titrated and antigen slides were prepared according to GB IFA． The best working concentration of IgG-FITC was titrated and the specificity，sensitivity and stability were tested． Results BSC-1 cells were selected as Japanese encephalitis virus ( JEV) -sensitive cells． The TCID50 of the virus was 10 － 5. 8·mL － 1． The best working concentration of the IgG-FITC was 1: 100. There was no cross-reactivity with ectromelia virus and mouse hepatitis virus． The tests of sensitivity and stability showed that the detection sensitivity was 1: 1280 at different time points． The lowest detected virus titer was 10 － 4. 1·mL － 1． Conclusions The established IFA method is good in sensitivity，specificity and stability，and can be used in detecting the Reo3 in animal-derived biological materials and biological products for human use．