Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, caused a global Coronavirus Disease 2019 (COVID-19) pandemic, and continuously threatens public health worldwide. The surface spike (S) protein of SARS-CoV-2 serves as a critical target for development of effective vaccines and therapeutic antibodies. Nevertheless, the S protein mutates rapidly, particularly in its receptor-binding domain (RBD) region, resulting in at least five variants of concerns, thereby making the vaccines targeting the S/RBD of previous variants less effective against the recent Omicron variant and/or its subvariants. Effective treatments, such as nanobodies, have displayed therapeutic potential against SARS-CoV-2 infection. Nanobodies may target conserved epitopes across different variants and/or subvariants and can access hidden epitopes that are typically not achievable by conventional antibodies. In this study, nanobodies targeting S protein of SARS-CoV-2 Omicron variant were isolated from immunized nanobody phage display libraries, and their activities, including binding affinity; and stability, were analyzed using a series of in vitro assays.