PD-1/PD-L1 pathway blocking with antibodies offers a vital and efficient therapeutic strategy to restore T cell-associated antitumor immunity and treats a variety of cancers in clinic. Nanobodies (Nbs) give several advantages over conventional monoclonal antibodies such as size, solubility, stability and costs. Additionally, P.?pastoris is a suitable host for Nb production. Herein, we aim to produce and evaluate anti-PD-1 Nb derived from the P.?pastoris. Our findings indicated that we successfully established the Nbs phage-displayed library against PD-1 with qualified library capacity and insert ratio. Anti-PD-1 Nb Nb97 was screened through PE-ELISA and flow cytometry. To extend half-life of Nb97, we contracted pPICZɑA-Nb97-Nb97-HSA recombination vector, which was then transformed into the system of P.?pastoris X-33. The yield of purified Nb97-Nb97-Human serum albumin (HSA) fused protein (MY2935) reached to 2.3?g/L after 147?h of fermentation. Meanwhile, the blocking effect of MY2935 is similar to that of MY2626 (humanized Nb97-Fc), and MY2935 showed better performance on stimulating the immune function through PD-1 reporter assay. Hence, P.?pastoris X-33 expressing and secreting functional anti-PD-1 Nb-HSA fusion protein might be a system of high yield and low cost.,Copyright ? 2019. Published by Elsevier Inc.