Objective To explore the fast diagnostics of activated infection of human herpes virus type 6 (HHV-6) with IEA/ex3( an antibody specific for an immediate early protein encoded by variant B HHV-6 U90 gene)in infants with fever and rash . Methods To collect the 55 samples of peripheral blood mononuclear cells (PBMCs) from clinical infants whose having a fever and rash, age are below 2 years old.The immediate early antigen (IEA) of HHV-6 were analyzed with IEA/ex3 in PBMCs by indirect immunofluorescence then using confocal microscope to observe, and using fluoromicroscope to observe after the PBMCs of 44 samples were incubated with cord blood cell at 37℃ in a humidified 5% CO2 atmosphere for 48 hours. The virus was isolated from the PBMCs. Results The conformation rate of the HHV-6 IEA using confocal microscope to observe and virus isolation was 92.73%, sensitivity was 90.32%, specificity was 95.83% and it was respectively 87.50%, 77.27%, 100% using fluoromicroscope to observe after the PBMCs were incubated for 48 hours. Conclusion The study suggested that detection of HHV-6B IEA in PBMCs using confocal microscope to observe after the PBMCs were incubated for 48 hours can fast diagnose HHV-6 activated infection of infant with fever and rash than technique of virus isolation.