A sensitive, specific and rapid method for the detection of three different kinds of plant viruses including tomato ringspot virus (ToRSV), bean pod mottle virus (BPMV) and arabis mosaic virus (ArMV) was demonstrated using novel upconversion nanoparticles (UCNPs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic nanoparticles (MNPs, ~100 nm) were coated with different antibodies were employed to capture and enrich the target viruses. Then antibody-conjugated UCNPs as signal probes were added to form sandwich complexes. This was followed by a fluorescence measurement using a 980 nm laser. This method not only avoids the difficulty in simultaneous detection of multiple independent organic fluorphores that require distinct excitation wavelengths, and auto-fluorescence of biological samples due to the higher-energy excitation of QDs but also amplifies detection signal by UCNPs-tags together with easy separation of samples by magnetic forces, demonstrating the potential to be used for detecting virus in the field of environmental safety and other fields.