Abstract: An anti-PD-1 nano-antibody capable of blocking a binding interaction between PD-1 and a ligand thereof, PD-L1. Disclosed are the nano-antibody, a nucleotide sequence encoding the nano-antibody, a corresponding expression vector, a host cell expressing the nano-antibody, and a method of producing the nano-antibody. Also disclosed is a humanized PD-1 nano-antibody sequence. The humanized nano-antibody can block the binding interaction between PD-1 and the ligand thereof, PD-L1 while still retaining a high affinity.