A lateral flow biosensor based on immunoassay has been developed for the detection of human stem cells for the first time. Antibody specific for a human stem cell surface antigen, SSEA-4, is coated onto gold nanoparticles, whereas antibody against another human pluripotent stem cell surface antigen, SSEA-3, is immobilized on the test zone of the NC membrane. Target cells bind to the antibody coated on the gold nanoparticles to form nanoparticles-stem cell complexes, and the complexes are then captured by another antibody immobilized on the test zone to form a red line for visual detection. This biosensor has been successfully applied to human embryonic stem cells and induced pluripotent stem cells. It is capable of detecting a minimum of 10,000 human embryonic stem cells by the naked eye and 7000 cells with a portable strip reader within 20 min. This approach has also shown excellent specificity to distinguish other types of cells. The biosensor shows great promise for specific and handy detection of human pluripotent stem cells.