Significance Developing molecules capable of binding to SARS-CoV-2 spike protein and inhibiting viral infection is of utmost importance for the detection and therapy of COVID-19. We have developed and engineered a serum-stable RNA aptamer specific for SARS-CoV-2 spike protein. We further show that scaffolding three aptamers together increases the binding efficiency to the low picomolar range and enables very efficient neutralization of SARS-CoV-2 infection in cells. The aptamer also shows high affinity for spike protein from variants of concern. Due to its small size and chemical stability, our aptamer holds potential as an alternative to antibodies and nanobodies targeting spike protein.