A nanobody and its application based on SARS-CoV-2 S protein are provided, and the present disclosure relates to biomedical technology. The present disclosure chooses the Spike S1+S2 ECD of SARS-CoV-2 as a target, and screens the nanobody against of SARS-CoV-2 by using a nanobody library. After an ELISA test, the Spike S1+S2 ECD target of SARS-CoV-2 can be specifically identified while a SPIKE RBD target is identified, and a binding signal is relatively strong. The corresponding nanobody sequence is constructed into a prokaryotic expression vector for expression and purification to express the target nanobody successfully. After the purification, the purity is greater than 90%. The ELISA test of VHH nanobody showed that the purified nanobody has higher affinity to the two targets.