Objective To screen the sequence of nanobodies against human CD20, and obtain anti-CD20-human IgG Fc nanobodies with high affinity and specificity. Methods Based on the naive phage display library, 4 rounds of liquid affinity screening were performed using biotinylated CD20 antigen as the target, and positive clones were identified by ELISA. Prokaryotic expression vector CD20-IgG Fc/pCZN1 was constructed and transformed into E.coli Arctic Express, and the expression of the recombinant protein was induced by IPTG at low temperature and purified by Ni column. The purified product was identified by ELISA and Western blot analysis. Results The specific CD20 nanobody showed good repeatability and hydrophilicity. The purity of anti-CD20-human IgG Fc nanobodies was higher than 85%. ELISA indicated that anti-CD20-human IgG Fc nanobodies had high affinity with CD20 antigen, and Western blot analysis demonstrated they could specifically recognize CD20 antigen. Conclusion The sequence of anti-CD20 nanobody was successfully obtained using the naive phage nanobody library. The purified anti-CD20-human IgG Fc nanobody has high affinity and specificity.